Riboflavin mutants as vaccines against Actinobacillus pleuropneumoniae

ABSTRACT

Described is a vaccine against Actinobacillus pleuropneumoniae (APP) comprising genetically defined biochemically attenuated mutant of APP that requires riboflavin and is attenuated in vivo.

CROSS REFERENCE TO RELATED CASES

This application includes the disclosure in provisional patent application serial No. 60/007,764 filed Nov. 30, 1995, for Characterization of Actinobacillus pleuropneumoniae Riboflavin Biosynthesis Genes, hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to vaccines and in particular, live vaccines against Actinobacillus pleuropneumoniae (APP) and related bacterial pathogens. The invention is also concerned with recombinant techniques for preparing such a vaccine.

BACKGROUND OF THE INVENTION

An organism known as Actinobacillus pleuropneumoniae (APP) is a gram negative coccobacillus organism that is found in the pig and causes pneumonia in the pig.

This disease is characteristically an acute necrotizing hemorrhagic bronchopneumonia, with accompanying fibrinous pleuritis (Fenwick, B. and S. Henry. 1994. Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 204:1334-1340)(Sebunya, T.N.K. and J.R. Saunders. 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182:1331-1337). Porcine pleuropneumonia is an economically devastating, severe and often fatal disease with clinical courses ranging from hyperacute to chronic infection (Fenwick, B. and S. Henry. 1994. Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 204:1334-1340)(Hunneman, W. A. 1986. Incidence, economic effects, and control of Haemophilus pleuropneumoniae infections in pigs. Vet. Quarterly 8:83-87). The existence of at least twelve antigenically distinct capsular serotypes (Perry, M. B., E. Altman, J. -R. Brisson, L. M. Beynon, and J. C. Richards. 1990. Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus pleuropneumoniae strains. Serodiag. Immunother. Infect. Dis. 4:299-308) has made development of a cross-protective vaccine difficult. Killed whole cell bacterins provide at best serotype-specific protection (Nielsen, R. 1984. Haemophilus pleuropneumoniae serotypes--Cross protection experiments. Nord. Vet. Med. 36:221-234)(Nielsen, R. 1976. Pleuropneumonia of swine caused by Haemophilus pleuropneumoniae. Studies on the protection obtained by vaccination. Nord. Vet. Med. 28:337-338) (Rosendal, S., D. S. Carpenter, W. R. Mitchell, and M. R. Wilson. 1981. Vaccination against pleuropneumonia in pigs caused by Haemophilus pleuropneumoniae. Can. Vet. J. 22:34-35)(Thacker, B. J., and M. H. Mulks. 1988. Evaluation of commercial Haemophilus pleuropneumoniae vaccines. Proc. Int. Pig Vet. Soc. 10:87). In contrast, natural or experimental infection with a highly virulent serotype of A. pleuropneumoniae elicits protection against reinfection with any serotype (Nielsen, R. 1979. Haemophilus parahaemolyticus serotypes: pathogenicity and cross immunity. Nord. Vet. Med. 31:407-413)(Nielsen, R. 1984. Haemophilus pleuropneumoniae serotypes--Cross protection experiments. Nord. Vet. Med. 36:221-234)(Nielsen, R. 1974. Serological and immunological studies of pleuropneumonia of swine caused by Haemophilus parahaemolyticus. Acta Vet. Scand. 15:80-89). In several recent studies, attenuated strains of A. pleuropneumoniae produced by chemical mutagenesis, serial passage, or other undefined spontaneous mutation have been tested as live vaccines, with promising results (Inzana, T. J., J. Todd, and H. P. Veit. 1993. Safety, stability and efficacy of nonencapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines. Infect. Immun. 61:1682-1686) (Paltineanu, D., R. Pambucol, E. Tirziu, and I. Scobercea. 1992. Swine infectious pleuropneumonia: Aerosol vaccination with a live attenuated vaccine. Proc. Int. Pig. Vet. Soc. 12:214) (Utrera, V., C. Pijoan, and T. Molitor. 1992. Evaluation of the immunity induced in pigs after infection with a low virulence strain of A. pleuropneumoniae serotype 1. Proc. Int. Pig Vet. Soc. 12:213). However, the use of live vaccines in the field is problematic, particularly when the attenuating lesion in the vaccine strain has not been genetically defined. A well-defined mutation that prevents reversion to wild-type would be extremely desirable for the development of a live attenuated vaccine against Actinobacillus pleuropneumoniae.

A variety of mutations in biosynthetic pathways are known to be attenuating in other organisms. Lesions in aro(Hoiseth S. K. and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (london). 291: 238-239)(Homchampa, P., R. A. Strugnell and B. Adler. 1992. Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant. Mol. Microbiol. 6: 3585-3593)(Homchampa, P., R. A. Strugnell and B. Adler. 1994. Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica. Vet. Microbiol. 42:35-44) (Karnell, A., P. D. Cam, N. Verma and A. A. Lindberg. 1993. AroD deleteion attenuates Shigella flexneri strain 2457T and makes it a safe and efficacious oral vaccine in monkeys. Vaccine 8:830-836.) (Lindberg, A. A., A. Karnell, B. A. D. Stocker, S. Katakura, H. Sweiha and F. P. Reinholt. 1988. Development of an auxotrophic oral live Shigella flexneri vaccine. Vaccine 6:146-150)(O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423)(Vaughan, L. M., P. R. Smith, and T. J. Foster. 1993. An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the Salmonid disease furunculosis. Infect. Immun. 61:2172-2181), pur (O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423)(Sigwart, D. F., B. A. D. Stocker, and J. D. Clements. 1989. Effect of a purA mutation on the efficacy of Salmonella live vaccine vectors. Infect. Immun. 57:1858-1861), and thy (Ahmed, Z. U., M. R. Sarker, and D. A. Sack. 1990. Protection of adult rabbits and monkeys from lethal shigellosis by oral immunization with a thymine-requiring and temperature-sensitive mutant of Shigella flexneri Y. Vaccine. 8:153-158) loci, which affect the biosynthesis of aromatic amino acids, purines, and thymine, respectively, are attenuating because they eliminate the ability of the bacterium to synthesize critical compounds that are not readily available within mammalian hosts. For example, aro mutants of Salmonella and Shigella species have been shown to be attenuated in their natural hosts (Hoiseth S. K. and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (london). 291: 238-239)(Homchampa, P., R. A. Strugnell and B. Adler. 1992. Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant. Mol. Microbiol. 6: 3585-3593)(Homchampa, P., R. A. Strugnell and B. Adler. 1994. Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica. Vet. Microbiol. 42:35-44)(Karnell, A., P. D. Cam, N. Verma and A. A. Lindberg. 1993. AroD deletion attenuates Shigella flexneri strain 2457T and makes it a safe and efficacious oral vaccine in monkeys. Vaccine 8:830-836)(Lindberg, A. A., A. Karnell, B. A. D. Stocker, S. Katakura, H. Sweiha and F. P. Reinholt. 1988. Development of an auxotrophic oral live Shigella flexneri vaccine. Vaccine 6:146-150) (O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423). Lesions that affect the biosynthesis of LPS (Collins, L. V., S. Attridge, and J. Hackett. 1991. Mutations at rfc or pmi attenuate Salmonella typhimurium virulence for mice. Infect. Immun. 59:1079-1085)(Nnalue, N. A., and B. A. D. Stocker. 1987. Tests of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella cholerasuis. Infect. Immun. 55:955-962) and of cyclic AMP (Kelly, S. M., B. A. Bosecker and R. Curtiss III. 1992. Characterization and protective properties of attenuated mutants of Salmonella cholerasuis. Infect. Immun. 60:4881-4890) (Tacket, C. I., D. M. Hone, R. Curtiss III, S. M. Kelly, G. Losonsky, L. Guers. A. M. Harris, R. Edelman. M. M. Levine. 1992. Comparison of the safety and immunogenicity of ΔaroC ΔaroD and ΔcyaΔcrp Salmonella typhi strains in adult volunteers. Infect. Immun. 60:536-541) have also been shown to be attenuating in Salmonella species. It is important to note that not all attenuating mutations are good vaccine candidates in different organisms because some attenuating mutations result in poor persistence and immunogenicity (O'Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423)(Sigwart, D. F., B. A. D. Stocker, and J. D. Clements. 1989. Effect of a purA mutation on the efficacy of Salmonella live vaccine vectors. Infect. Immun. 57:1858-1861).

Riboflavin (vitamin B2), a precursor of the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), is essential for basic metabolism. It is synthesized by plants and by most microorganisms but not by higher animals (Bacher, A. 1991. Biosynthesis of flavins. p. 215-59. In F. Muller (ed.), Chemistry and Biochemistry of Flavins, Vol. 1. Chemical Rubber Company, Boca Raton, Fla.). Many pathogenic bacteria are apparently unable to utilize flavins from their environment and are entirely dependent on endogenous production of riboflavin (Schott, K., J. Kellerman, F. Lottspeich and A. Bacher. 1990. Riboflavin synthases of Bacillus subtilis: purification and amino acid sequence of the α-subunit. J. Biol.Chem. 265:4204-4209). Even with the ability to utilize exogenous riboflavin, there may not be enough of the vitamin present in mammalian host tissues to permit growth, particularly not in sites devoid of normal bacterial flora.

Vaccines are preparations used to prevent specific diseases in animals by inducing immunity. This is accomplished by exposing a patient to an antigen from an agent capable of causing a particular disease which, in turn, causes the immune system of the patient to produce large quantities of antibody. The presence of the antibody in the patient's blood protects the patient from a later attack by the disease-causing agent. Vaccines may either be composed of subunits of the agent, or the live or killed agent itself. If a live vaccine is to be used, its virulence must be attenuated in some way; otherwise, the vaccine will cause the disease it is intended to protect against. See U.S. Pat. No. 5,429,818, Col. 1.

Most current vaccines against APP are killed whole cell bacterins, that is, whole bacterial cells killed by heat treatment or formalinization, suspended in an adjuvant solution. Some alternative ways of attempting to develop vaccines against APP are the use of subunit vaccines and the use of non-encapsulated mutants.

The use of a protease lysate of the outer membrane of A. pleuropneumoniae cells as a vaccine against APP infection is described in U.S Pat. No. 5,332,572.

The use of extracellular proteins and/or hemolysins from APP as vaccines against APP infection is described in U.S. Pat. Nos. 5,254,340, WO Patent No. 9409821, EP No. 595,188, CA 2045950, and EP No. 453,024.

The use of non-encapsulated mutants of APP is described in U.S. Pat. No. 5,429,818. It disclosed that the capsule of such bacteria is required for virulence. Therefore, the preparation of a mutant of APP that was a non-capsulated mutant was described as a vaccine.

A method of administering vaccines to pigs by a transthoracic intrapulmonary immunization is described in U.S. Pat. No. 5,456,914.

A vaccine for the immunization of an individual against Salmonella choleraesuis utilizing derivatives that are incapable of producing functional adenylate cyclase and/or cyclic AMP receptor protein is described in U.S. Pat. No. 5,468,485. The avirulent S. choleraesuis was made avirulent by an inactivating mutation in a cya gene and an inactivating mutation in a crp gene. Similar techniques are described in other bacteria in U.S. Pat. Nos. 5,424,065; 5,389,386; 5,387,744 and 4,888,170.

To protect animals from lung disease, it is needed to achieve a sufficiently high level of antibodies, particularly IgA antibodies, in the lungs to prevent adherence of invading microorganisms to mucosal surfaces and neutralize potentially damaging virulence factors. Antibodies in the patient's serum or at the mucosal surfaces can be important to protection. One of the reasons for using a live vaccine instead of a killed whole cell bacterin is that a live vaccine, given intranasally or orally, can induce specific local secretory antibody in the secretions that cover mucosal surfaces. This local antibody is often quite helpful for protection against diseases that infect at or through mucosal surfaces.

None of the patents pertain to a recombinant technique for a relatively convenient method for obtaining genetically defined mutants for use in a vaccine against APP.

It is believed that a mutation in a critical biosynthetic pathway which limits growth in vivo but does not otherwise alter expression of important antigens such as capsular polysaccharide, lipopolysaccharide and extracellular toxins, could produce an attenuated vaccine strain capable of inducing cross-protective immunity against A. pleuropneumoniae.

It is believed that riboflavin biosynthesis would be essential for survival of A. pleuropneumoniae in vivo, and that mutations in the riboflavin biosynthetic pathway would be attenuating due to the scarcity of riboflavin present on the mucosal surfaces of the respiratory tract.

It is an object of the present invention to describe the use of mutations in the riboflavin biosynthetic pathway to construct attenuated strains of pathogenic bacteria for use as live vaccines, with a riboflavin-requiring mutant of APP used as a specific example.

It is an object of the present invention to describe a live vaccine against APP utilizing a riboflavin mutation in the APP genome.

SUMMARY OF THE INVENTION

Described is a live vaccine against bacterial pathogens comprising a recombinant riboflavin-requiring mutant having a mutation that inactivates riboflavin biosynthesis therein. In particular, this includes bacterial pathogens in the family Pasteurellaceae, which include animal pathogens as Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica and Pasteurella multocida, as well as human pathogens Haemophilus influenzae and Haemophilus ducreyi.

Also described is a live vaccine against Actinobacillus pleuropneumoniae (APP) comprising a recombinant APP having an attenuating inactivating mutation therein.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIG. 1 describes a proposed a metabolic pathway for bacterial riboflavin synthesis.

FIG. 2 is a physical map of a plasmid contruct pTF-10, which comprises a 5.2 Kb fragment of A. pleuropneumoniae chromosomal DNA, including the APP riboflavin biosynthetic operon, cloned into the vector pUC19.

FIG. 3 shows the absorbance spectra of aqueous solutions at neutral pH (Panel A) and acidified aqueous solutions (Panel B) of the product excreted into the growth medium by E. coli DH5α/pTF10 (solid line) and of a standard riboflavin preparation (dotted line).

FIG. 4 shows the complete nucleotide sequence of APP ribGBAH operon and flanking regions and the predicted amino acid sequences of the encoded proteins.

FIG. 5 shows the complementation of E. coli riboflavin-requiring mutants by cloned APP rib genes. A physical map for the APP ribGBAH genes is shown as well as several deletions.

FIG. 6 shows a minicell analysis of the proteins encoded by pTF10 and its deletions.

FIG. 7 shows the construction of pTF67a, a suicide delivery vector containing a portion of the APP rib operon with a part of ribB and all of ribA deleted and replaced with a gene cassette encoding resistance to the antibiotic kanamycin.

FIG. 8A is an analysis of rib-transconjugants of A. pleuropeneumoniae serotype 1.

FIG. 8B is a southern blot analysis of chromosomal DNA from A. pleuropneumoniae serotype 1 rib mutants.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present application pertains to the development of attenuated mutants of the pathogenic bacterium A. pleuropneumoniae which contain mutations in the genome, specifically in the genes encoding the enzymes involved in the biosynthesis of riboflavin. By "mutation" is meant not just a random selection of variations of the genome of APP but utilization of well known recombinant techniques for specifically modifying the genome of APP. Accordingly, therefore, it is desirable to ascertain the riboflavin biosynthesis genes of APP.

By "attenuated" is meant a reduction in the severity, virulence or vitality of the disease causing agent.

After determining the sequence and organization of the riboflavin genes, one is then able to modify APP by removing some or all of such genes, thereby attenuating the pathogen, i.e., making the pathogen avirulent.

After a strain of avirulent APP is obtained, it could then be utilized as a live vaccine. Described below are the detailed steps broadly outlined above.

IDENTIFYING, CLONING, AND SEQUENCING OF THE RIBOFLAVIN BIOSYNTHESIS GENES FROM APP

Cloning of riboflavin genes from APP is described in the paper entitled "Characterization of APP Riboflavin Biosynthesis Genes", Journal of Bacteriology, December, 1995, pages 7265-7270 by Fuller and Mulks. This is incorporated herein by reference.

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia (9,23,39). The disease is characteristically an acute necrotizing hemorrhagic bronchopneumonia, with accompanying fibrinous pleuritis (9,39). Pleuropneumonia is an economically devastating, severe and often fatal disease with clinical courses ranging from peracute to chronic infection (9,14). The existence of at least twelve antigenically distinct capsular serotypes (31) has made development of a cross-protective vaccine difficult. Killed whole cell bacterins provide at best serotype-specific protection (25,26,35,43). In contrast, natural or experimental infection with virulent APP frequently elicits protection against reinfection with any serotype (24,25,27). Avirulent strains of APP have been tested as live vaccines and have elicited cross-protective immunity against subsequent challenge (15,28,44). However, the use of live vaccines in the field is problematic, particularly when the attenuating lesions in the vaccine strain have not been genetically defined. Development of attenuated strains with defined biochemical mutations that limit growth in vivo and prevent reversion to wild type is a promising approach to improved vaccines against APP infection.

Riboflavin (vitamin B2), a precursor of the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), is essential for basic metabolism. It is synthesized by plants and by most microorganisms but not by higher animals (1). Many pathogenic bacteria are apparently unable to utilize flavins from their environment and are entirely dependent on endogenous production of riboflavin (38). Therefore, riboflavin biosynthesis may be essential for survival of pathogens in vivo, and mutations in the riboflavin biosynthetic pathway may be attenuating.

The proposed metabolic pathway for bacterial riboflavin synthesis shown in FIG. 1 begins with guanosine triphosphate (GTP ) as the precursor (for a review see reference 1). The most extensively studied system for bacterial riboflavin synthesis is Bacillus subtilis (for a review see reference 29). The B. subtilis riboflavin synthesis genes are located and coregulated in an operon structure (12) that consists of five open reading frames designated as ribG, rib B, rib A, ribH and ribT (19,29) . The ribGBAHT genes encode, respectively, a rib-specific deaminase; the α-subunit of riboflavin synthase (lumazine synthase); a bifunctional enzyme containing GTP cyclohydrase and 3,4-dihydroxy 2-butanone 4-phosphate synthase (DHBP) activities; the β-subunit of riboflavin synthase; and a rib-specific reductase (29). The complete sequence of the B. subtilis riboflavin operon has been determined in two individual laboratories (19,30). The B. subtilis structural ribGBAHT genes code for predicted proteins of 361 (MW 39,700), 215 (MW 23,600), 398 (MW 43,800), 154 (MW 16,900), and 124 (MW 13,600) amino acids in length (19, 29). Two functional promoters have been identified in the B. subtilis rib operon. The main promoter, P1, for which a transcriptional start site has been determined 294 base pairs (bps) upstream of ribG (12,30), is responsible for transcription of all five structural genes (12). Another promoter, P2, produces a secondary transcript encoding the last three genes of the operon, ribAHT (12). A possible third promoter has been postulated that would express ribH (7). In addition, the operon has been shown to be transcriptionally coregulated (30) by a transacting repressor, RibC (3,6), which acts at a regulatory site, ribO (3,20), upstream of ribG, apparently by a transcription terminationantitermination mechanism (29). The RibC repressor appears to respond to FMN and FAD, as well as to riboflavin and several of its biosynthetic intermediates, and regulates expression from both ribPl and ribP2 (4,20,29).

E. coli is the second most chemically characterized system for riboflavin synthesis. In contrast to B. subtilis, the rib genes of E. coli are scattered around the chromosome and are expressed constituitively (2,46). Rather than having a bifunctional ribA, E. coli has two separate genes, ribB and ribA, that encode the functions of 3,4-DHBP synthase (34) and GTP cyclohydrase II (33), respectively. ribB is homologous to the 5' end of B. subtilis ribA while ribA is homologous to the 3' end (33,34). E. coli genes with sequence homology to the B. subtilis ribG (42), ribH (42), and ribB genes have also been identified.

Identified herein is a fragment of APP serotype 5 chromosomal DNA that triggers overproduction of riboflavin when cloned in E. coli. Nucleotide sequence analysis demonstrated four open reading frames with significant identity and a similar operon arrangement to the ribGBAH genes from Bacillus subtilis.

MATERIALS AND METHODS

Bacterial strains and media. A. pleuropneumoniae ISU178, a serotype 5 strain, was cultured at 37° C. in brain heart infusion broth or agar (Difco Laboratories, Detroit, Mich.) containing 10 μg/ml nicotinamide adenine dinucleotide (NAD) (Sigma Chemical Company, St. Louis, Mo.). E. coli DH5-α (supE44), ΔlacU169, (.o slashed.80lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was used for construction of the APP genomic library. E. coli strain DS410 (azi-8, tonA2, minA1, minB2, rpsL135, xyl-7, mtl-2, thi-1, λ-) was used for minicell isolation and protein labeling experiments. E. coli ribA:Tn5 (BSV18), ribB:Tn5 (BSV11) and ribC:Tn5 (BSV13) mutants used for complementation studies were described by Bandrin et al (2) and are available from Barbara Bachmann (E. coli Genetic Stock Center, Yale University). E. coli strains were cultured in Luria-Bertani medium or in M9 (36) supplemented with 15 g/L NZ (amine (Sigma) and with riboflavin at 200 μg/mL when necessary. Ampicillin was added to 100 μg/ml for plasmid selection.

DNA manipulations. DNA modifying enzymes were supplied by Boehringer-Mannheim Biochemicals (Indianapolis, Ind.) and used according to manufacturer's specifications. Genomic and plasmid DNA preparations, gel electrophoresis, and E. coli transformation were all performed by conventional methods (36).

Cloning and sequencing. APP serotype 5 genomic DNA was digested with HindIII and fragments ranging in size from 4 to 7 kb were ligated into the HindIII site in the polylinker of the plasmid vector pUC19 (45). A recombinant plasmid, designated PTF10, which overproduced riboflavin was isolated from this library. Unidirectional nested deletions were constructed with exonuclease III and S1 nuclease digestion, using the Erase-a base system (Promega Corp., Madison, Wis.). Nucleotide sequencing was performed on alkali-denatured double-stranded DNA by the dideoxy chain-termination method of Sanger et al. (37) using the Sequenase 2.0 kit (U.S. Biochemical, Cleveland, Ohio) and ³⁵ S!dATP (adenosine triphosphate) (Amersham Corp., Arlington Heights, Ill.). Sequencing primers used included universal forward and reverse primers for pUC sequencing (U.S. Biochemicals), as well as several oligonucleotide primers designed from previously obtained sequence data. These internal primers were synthesized by the Michigan State University Macromolecular Structure Facility and included MM4 (5'-AAT-CCG-GCA-AAA-ATT-GAA-GGC-3') (Sequence ID No: 1), MM5 (5'-GCA-CCG-TGA-CGC-ACT-AAC-G-3') (Sequence ID No: 2), MM6 (5'-GCG-CCA-ATA-CTT-GCT-CAC-CG-3') (Sequence ID No: 3), MM9 (5'-GGT-TTC-TTT-ATT-CGT-ATG-CGG-3') (Sequence ID No: 4), MM10 (5'-TGA-AGA-AGT-CGG-CAA-ATT-GCT-C-3') (Sequence ID No: 5), MM11 (5'-CGG-ATT-GGG-ATT-CGT-CCA-GCC-3') (Sequence ID No: 6), MM13 (5'-GGC-GAC-ACG-ATT-GCG-GTG-3') (Sequence ID No: 7), MM14 (5'-GCC-AGT-TAG-TGC-AGA-CAG-CG-3') (Sequence ID No: 8), and MM38 (5'-CTC-ACC-GGT-TCC-TGC-CAA-ACC-3') (Sequence ID No: 9).

DNA sequences were analyzed using the GCG sequence analysis programs (11).

Mass spectroscopy. Positive and Negative Ion Fast Atom Bombardment (FAB) mass spectroscopy was performed at the Michigan State University Mass Spectroscopy Facility.

Quantification of riboflavin. Bacterial cells were pelleted in a microcentrifuge, and the absorbance at 445 nm of the culture supernatant was measured using a Beckman DU-7 spectrophotometer (Beckman Instruments, Fullerton, Calif.). The absorbance at 445 nm was multiplied by a factor of 31.3 to yield the riboflavin concentration in mg/liter (10).

Minicell Analysis. The minicell-producing E. coli strain DS410 (32) was transformed by calcium chloride/heat shock treatment with pUC19 or pTF rib clones. Transformant colonies which produced a large number of minicells were selected by microscopy. Cultures were grown overnight at 37° C. in 500 mL LB broth, and minicells were isolated by differential centrifugation followed by glass fiber filtration as described by Christen et al (8). Minicells were resuspended to an OD₅₉₀ of 0.5-1.0 in 200 μl labeling mix (22.0 ml M9 media, 20.0 ml 50 mM HEPES (N- 2-hydroxyethyl! piperazine)-N'-2 ethanesulfonic acid) pH 7.5, 2.5 ml of 20% glucose, 0.05 ml of 10 mg/ml adenine, 0.05 ml of 10 mg/ml pyridoxine, 5.0 ml of NEDA amino acid stock (21) lacking methionine and cysteine, and 0.2 ml of 10 mg/ml cycloserine-D) and incubated at 37° C. for 30 minutes. Trans-label ( ³⁵ S!methionine plus ³⁵ S!cysteine, ICN Biomedicals, Irvine, Calif.) was added to a final concentration of 22 μCi per reaction and cells were incubated at 37° C. for 1 hour. Total and TCA (trichloroacetic acid) precipitable counts were measured by liquid scintillation counting to determine amount of incorporation. Cells were pelleted in a microcentrifuge and washed with cold HEPES (50 mM,pH7.5) plus 10 mM methionine plus 10 mM cysteine. Labeled proteins (50,000 cpm/lane) were separated by discontinuous SDS-PAGE on a 12% polyacrylamide gel and were visualized by autoradiography on Kodak XAR-5 film.

Nucleotide sequence accession number. The nucleotide sequence of the A. pleuropneumoniae ribGBAH genes has been submitted to GenBank and assigned an accession number of: U27202.

RESULTS

Identification of a riboflavin producing clone. A genomic library of A. pleuropneumoniae serotype 5 DNA was constructed in pUC19 and transformed into E. coli DH5-α. A single clone, designated pTF10 (FIG. 2), containing a 5.2 kbp insert, was identified that produced a bright yellow extracellular, water-soluble compound that fluoresced under ultraviolet light. The compound was crudely purified by filtration through a 3000 Da cut off membrane filter (Amicon Corporation, Bedford, Mass.). Absorbance spectra of this compound in aqueous solution under neutral conditions showed absorbance peaks at 373 and 443 nm, which coalesced to a single peak at 388 nm under acidic conditions; these results compared well to a riboflavin standard (FIG. 3). Positive and negative ion fast atom bombardment mass spectroscopy indicated that the compound was a flavin (data not shown). Culture of E. coli DH5-α/pTF10 in M9 medium plus NZ amine plus 0.6% glucose yielded 10 mg riboflavin per liter in 24 hours.

Sequence of APP rib genes. The nucleotide sequence and corresponding predicted amino acid sequence of a 4312 bp region of the insert in pTF10 is shown in FIG. 4. Four open reading frames of 1232, 647, 1205, and 461 bp were observed that encoded proteins with predicted molecular masses of 45,438 Da, 23,403 Da, 44,739 Da and 16,042 Da, respectively. Based on homology with the riboflavin biosynthetic genes of B. subtilis (see below), these ORFs were designated ribG, ribB, ribA, and ribH, respectively. All four ORFs were preceded by potential ribosome binding sites (RBS), although the RBS upstream of ribG is not as strong as the other three. Production of riboflavin by this clone is not dependent on its orientation in pUC19 or on induction by IPTG, indicating that it is produced under the control of a native promoter included in the cloned DNA fragment. A consensus promoter sequence of the -35/-10 type (12) was identified within the sequenced region 224 bp upstream from the ribG start codon. A second potential consensus promoter was identified between the genes ribA and ribH. However, no consensus promoter was identified between ribB and ribA, as is found in B. subtilis. The ORF encoding ribH is followed by an inverted repeat stem-loop structure with a ΔG=-56.0, that may function as a rho-dependent transcriptional terminator (13)

Homology of APP rib genes. Predicted amino acid sequences of the APP RibGBAH proteins were compared with B. subtilis RibGBAH (19); E. coli RibA, RibB, RibC, RibG, and RibH (33,34,42); Photobacterium leiognathi RibI-III (17), Photobacterium phosphoreum RibI-IV (16), and Vibrio harveyi LuxH (41) proteins, using the GCG Gap program (Table 1). APP RibG showed 62-63% similarity to the RibG proteins from B. subtilis and E. coli. APP RibB showed 58-69% and APP RibH showed 69-83% similarity to homologous genes from B. subtilis, E. coli, and Photobacterium species. APP RibA showed 73% similarity to the entire RibA protein of B. subtilis and 61% to the RibII protein of P. leiognathi, both of which encode a bifunctional enzyme catalyzing two distinct steps in the riboflavin pathway. In addition, the carboxy terminal half of APP RibA, encompassing ˜200 amino acids, shows 59-63% similarity to E. coli RibB, and V. harveyi LuxH, which encode 3,4-DHBP synthase. The N-terminal region of APP RibA, encompassing the remaining ˜200 amino acids, shows 63-73% similarity to E. coli RibA and P. phosphoreum RibIV, which encode GTP cyclohydrase II.

                                      TABLE 1     __________________________________________________________________________     Percent similarity of amino acid sequences of riboflavin synthesis     proteins.sup.a     % Similarity with A. pleuropneumoniae             RibG    RibB    RibA    RibH             Compared                     Compared                             Compared                                     Compared     Bacterium             With %  With %  With %  With %     __________________________________________________________________________     B. subtilis             RibG 63 RibB 69 RibA 73 RibH 83     E. coli.sup.b             RibG 62 RibC 58 RibB 63 RibH 74                             RibA 73     H. influenzae.sup.c             RibG 58 RibC 60 RibB 65 RibE 75                             RibA 71     P. leiognathi                  NA RibI 64 RibII                                  61 RibIII                                          69     P. phosphoreum.sup.d                  NA RibI 63 RibII                                  59 RibIII                                          73                             RibIV                                  63     V. harveyi   NA      NA LuxH 59      NA     __________________________________________________________________________      .sup.a Identity is expressed in percent similarity as calculated by the      Genetics Computer Group NeedlemanWunsch algorithm (22). Proteins with      partial identify were compared with the entire appropriate A.      pleuropneumoniae Rib protein.      .sup.b E. coli RibB is homologous to the 5' end of A. pleuropneumoniae      RibA. E. coli RibA is homologous to the 3' end of A. pleuropneumoniae      RibA.      .sup.c H. influenzae RibB is homologous to the 5' end of A.      pleuropneumoniae RibA H. influenzae RibA is homologous to the 3' end of A      pleuropneumoniae RibA.      .sup.d P. phosphoreum RibIV is homologous to the 3' end of A.      pleuropneumoniae RibA.

Complementation of E. coli mutants. The original pTF10 clone and several deletion derivatives were tested for their abilities to complement ribA (GTP cyclohydrase II), ribB (3,4-DHBP synthase), and ribC (β-subunit of riboflavin synthase) mutations in E. coli (2) (FIG. 5) Complementation of the E. coli mutation was determined by restoration of the ability to grow on M9 minimal medium in the absence of riboflavin. Plasmids containing a complete copy of the APP ribB gene complemented the E. coli ribC mutation. Plasmids containing the 5' end of APP ribA complemented the E. coli ribB mutation. Plasmids containing a complete copy of APP ribA complemented both E. coli ribB and ribA mutations.

Minicell analysis. Plasmid pTF10 and its deletion derivatives were transformed into the minicell-producing E. coli strain DS410, and the proteins encoded by these plasmids were radioactively labeled, separated by SDS-PAGE, and visualized by autoradiography. Compared with the pUC19 vector, plasmid pTF10 shows four unique proteins with apparent molecular masses of 45 kDa, 27.7 kDa, 43.7 kDa, and 14.8 kDa (FIG. 6), which correspond well with the sizes predicted for the RibG, RibB, RibA, and RibH proteins by amino acid sequence data. The RibG protein did not appear to be as strongly expressed as RibB, RibA, and RibH. Analysis of proteins encoded by plasmid pTF19 (FIG. 5), which contains no ribH and a slightly truncated ribA gene, eliminates the 14.8 kDa protein (RibH) and truncates the 43.7 kDa protein (RibA) to 42.5 kDa (FIG. 6). Plasmid pTF12 (FIG. 5), which does not contain ribb, ribA, or ribH genes, does not express the 27.7, 43.7, or 14.8 kDa proteins (data not shown).

Described above is the identification, cloning and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in E. coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from both E. coli and Bacillus subtilis. The genes have been designated APP ribGBAH due to their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon.

The DNA sequence data, complementation, and minicell analysis strongly suggest that the four rib genes are transcribed from a single APP promoter upstream of the ribG gene. This promoter, among the first described for housekeeping genes in APP, is a good match for an E. coli consensus 35/-10 promoter. There is a 4 of 6 bp match at the -35 region, a 17 bp interval, a 4 of 6 bp match at the -10 region, an 8 bp interval, and a CAT box at the -1/+1 site. There is also a second potential promoter located between ribA and ribH, although it is not clear whether this promoter is functional.

Biosynthesis of riboflavin by APP appears to be more similar to that in the gram-positive bacterium B. subtilis than in the gram-negative bacterium E. coli. First, APP rib genes are arranged in an operon similar to that seen in B. subtilis, rather than scattered throughout the chromosome as is found in E. coli. However, the B. subtilis rib operon contains a fifth gene, ribT, that is proposed to mediate the third step in riboflavin biosynthesis; it is unlikely that a ribT homologue is present as part of the operon in APP because of the presence of a strong inverted repeat following ribH and the lack of a likely reading frame downstream. Second, APP contains a ribA gene that encodes a bifunctional enzyme with both GTP cyclohydrase II and DHPB synthase activities, as is found in B. subtilis; E. coli has two genes, ribA and ribB, that encode these two enzymes separately. Finally, the APP riboflavin biosynthetic enzymes are more similar at the amino acid level to the enzymes of B. subtilis than to those of E. coli, although alignment of the proteins from all three sources shows highly conserved sequences (data not shown).

It should be noted that in three bioluminescent species from the family Vibrionaceae, Vibrio harveyi, Photobacterium leiognathi, and P. phosphoreum, riboflavin biosynthesis genes have been shown to be closely linked to the lux operon (10, 11, 41). FMNH₂ is the substrate for the light-emitting reaction, and therefore an increase in bioluminescence requires an increased supply of the cofactor. Since riboflavin is the precursor for FMN, linkage and possibly coordinate regulation of lux and rib genes may facilitate the expression of bioluminescence in these bacteria.

The recombinant E. coli DH5-α containing plasmid pTF10 showed a marked increase in extracellular riboflavin production, most likely due to the lack of regulation (40) and high copy number of the cloned synthetic genes (45). Although the APP rib operon is similar in structure to that of B. subtilis, it is not yet known whether the genes are tightly regulated in APP by a repressor similar to B. subtilis RibC, or whether they are constituitively expressed as appears to be true in E. coli (33). It is believed APP must synthesize riboflavin to meet its own metabolic demands during infection, since riboflavin is not synthesized by mammals and therefore is not likely to be freely available to APP within its porcine host.

ATTENUATION OF RIB- MUTANTS OF A. PLEUROPNEUMONIAE

Applicants have constructed deletion-disruption riboflavin-requiring mutants of A. pleuropneumoniae serotypes 1 and 5.

Applicants have conducted experiments to confirm that the Rib- APP mutants constructed are attenuated in swine.

In a preliminary experiment, seven 8-to-10 week old pigs were used. Three pigs were infected endobronchially with Nal^(R) (resistant to the antibiotic malidixic acid) derivatives of wild type virulent APP-1 or APP-5; three were infected with APP-5 Rib- mutants; and one was used as an uninfected control. The APP strains, dosages used for infection, and results are summarized below in Table 2. Animals were euthanized when clinical signs became severe or at 12 hours post infection. The animals were necropsied and the lungs examined for gross pathology and histopathology, and lungs were cultured to recover APP.

                  TABLE 2     ______________________________________     Summary of clinical signs, gross pathology, and histopathology     seen in pigs challenged with either wild type APP serotype 1 or     5 or Rib- mutants of App Serotype 5     APP Strain and     Description Dosage    Results     ______________________________________     AP225: APP-1, Nal.sup.R                 2 × 10.sup.8 cfu                           Died, <4 hrs; 4+ peracute                           hemorrhagic pneumonia lesions     AP227: APP-5, Nal.sup.R                 1 × 10.sup.9 cfu                           Died, <4 hrs: 4+ peracute                           hemorrhagic pneumonia lesions     AP228: APP-5, Nal.sup.R                 1 × 10.sup.9 cfu                           Died, <4 hrs: 4+ peracute                           hemorrhagic pneumonia lesions     AP229: APP-5, Nal.sup.R,,                 1 × 10.sup.9 cfu                           Mild clinical signs; 1+ mild     Km.sup.R, Rib-        pneumonia lesions     AP230: APP-5, Nal.sup.R,,                 1 × 10.sup.9 cfu                           Mild clinical signs; 1+ mild     Km.sup.R, Rib-        pneumonia lesions     AP231: APP-5, Nal.sup.R,,                 1 × 10.sup.9 cfu                           Mild clinical signs; 1+ mild     Km.sup.R, Rib-        pneumonia lesions     None        --        1+ mild                           pneumonia lesions (mycoplasma?)     ______________________________________

Note that the dosage used in all of these animals was about 200 times the LD₅₀ (50% lethal dose, or the dose that will kill 50% of the animals exposed) for the wt (wild type) APP parent strains. The Nal^(R) derivatives of the wild type) parent strains retained virulence, triggering severe fibrinosuppurative hemorrhagic pneumonia and death within 4 hours. The Rib- mutants caused minimal clinical signs (increased respiration rate and slight fever) and at most mild signs of pneumonia, including some consolidation but no hemorrhagic necrosis, as compared to the uninfected control. These were not SPF (specific pathogen free) pigs, and there were histologic lesions suggestive of mild mycoplasma infection, in all of the pigs, including the uninfected control (Table 2).

Described below is the construction of a deletion-disruption riboflavin mutant of A. pleuropneumoniae serotype 1 (APP-1) and detailed analysis of the attenuation of this APP-1 Rib- mutant in vivo in swine.

MATERIALS AND METHODS

Bacterial strains and media. The bacterial strains and plasmids used in this study are listed in Table 1. A. pleuropneumoniae strains were cultured at 37° C. in either brain heart infusion (BHI), heart infusion (HI), or tryptic soy agar (TSA) (Difco Laboratories, Detroit, Mich.) containing 10 μg/ml NAD (V factor) (Sigma Chemical Company, St. Louis, Mo.). Riboflavin (Sigma) was added to a final concentration of 200 μg/ml when needed. E. coli strains were cultured in Luria-Bertani medium. Ampicillin was added to 100 μg/ml and kanamycin to 50 μg/ml for plasmid selection in E. coli strains. For A. pleuropneumoniae strains, 50 μg/ml kanamycin sulfate and 25 μg/ml nalidixic acid were added as required, except for selection after matings which were performed with 100 μg/ml kanamycin sulfate and 50 μg/ml nalidixic acid.

DNA manipulations. DNA modifying enzymes were supplied by Boehringer-Mannheim Biochemicals (Indianapolis, Ind.) and used according to the manufacturer's specifications. Genomic DNA was prepared according to the lysis/proteinase K method of the Gene Fusion Manual (Silhavy, T. J. 1984. DNA extraction from bacterial cells. p. 137-139. In Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.34). Plasmid DNA preparations, agarose gel electrophoresis, and E. coli transformation were all performed by conventional methods (Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).

Filter mating targeted mutagenesis. Filter mating between E. coli S17-1 (λpir) /pTF67A and AP225 was performed according to the protocol of Mulks and Buysse (Mulks, M. H. and J. M. Buysse. 1995. A targeted mutagenesis system for Actinobacillus pleuropneumoniae. Gene 165:61-66). Briefly, bacterial cultures were grown overnight at 37° C. Equal cell numbers of donor and recipient cultures, as determined by optical density at 520 nm, were added to 5 ml 10 mM MgSO₄ and the bacteria pelleted by centrifugation. The pellet containing the cell mating mixture, resuspended in 100 μl of 10 mM MgSO₄, was plated on a sterile filter on BHIV+riboflavin agar and incubated for 3 h at 37° C. Cells were washed from the filter in sterile phosphate buffered saline (pH 7.4), centrifuged, resuspended in 400 μl BHIV broth and plated in 100 μl aliquots on BHIV containing riboflavin, kanamycin, and nalidixic acid. Kanamycin and nalidixic acid resistant colonies were selected from filter mating plates and screened for riboflavin auxotrophy by replica plating onto TSAV, observing for inability to grow in the absence of added riboflavin.

Southern Analysis of Transconjugants. Chromosomal DNA and plasmid controls were digested with the appropriate restriction enzymes and the DNA fragments were separated on an 0.7% ultrapure agarose gel in TAE buffer. Southern blots were performed as described by Sambrook et al (Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). DNA probes were labeled with digoxygenin by random priming using the Genius V. 3.0 kit from Boehringer Mannheim. Probes included the 5.2 Kb insert from pTF10 containing the intact riboflavin operon from AP106 (Rib), the 1.4 Kb ClaI/NdeI fragment deleted from the riboflavin operon in the construction of pTF67a (R.Del.), the 1.2 Kb kanamycin cassette from pUC4K (Km) and the intact plasmid pGP704 (pGP704). Hybridization was carried out in 50% formamide at 42° C. for 16 h. Blots were washed twice in 2×SSC/0.1% SDS for 15 min at room temperature, then twice in 0.1×SSC/0.1% SDS for 30 min at 65° C. Blots were developed with alkaline phosphatase-conjugated anti-digoxygenin and calorimetric substrate (Boehringer Mannheim) according to the manufacturer's instructions.

Phenotypic analysis of mutant strains. Whole cell lysates and supernatants of AP100, AP225 (Nal^(R)), and AP233 (Km^(R), Nal^(R), Rib-) were prepared from overnight cultures grown in HIV+5 mM CaCl₂ +appropriate antibiotics. Cells were separated by microcentrifugation and resuspended in SDS-PAGE sample buffer (Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685). The culture supernatant was precipitated with an equal volume of 20% trichloroacetic acid (TCA) and resuspended in SDS-PAGE (sodium dodecyl sulfate--polyacrylamide gel electrophoresis) sample buffer. Cellular polysaccharides, including lipopolysaccharide (LPS) and capsular polysaccharide, were prepared according to the cell lysis/proteinase K method of Kimura et al (Kimura, A. and E. J. Hansen. 1986. Antigenic and phenotypic variations of Haemophilus influenzae type B lipopolysaccharide and their relationship to virulence. Infect. Immun. 51:69-79). All samples were analyzed on a 0.125% SDS-12% acrylamide gel using a discontinuous buffer system (Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685). Samples were transferred to nitrocellulose according to standard protocols (Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) and probed with convalescent serum from a pig infected with A. pleuropneumoniae serotype 1. Antigen-antibody complexes were detected with horseradish peroxidase-conjugated protein A (Boehringer Mannheim) and the colorimetric substrate 4-chloro-naphthol (BioRad, Hercules, Calif.).

Production of serotype-specific capsular polysaccharide was measured by coagglutination assay using hyperimmune rabbit anti-sera complexed to Staphylococcus aureus whole cells (Jolie, R. A. V., M. H. Mulks, and B. J. Thacker. 1994. Antigenic differences within Actinobacillus pleuropneumoniae serotype 1. Vet. Microbiol. 38:329-349).

Electroporation of A. pleuropneumoniae. AP233 was grown in 100 ml BHIV with riboflavin at 37° C., with shaking at 150 RPM, to an OD₅₂₀ of 0.7. Cells were chilled on ice and centrifuged at 5,000×g at 4° C. for 10 min. Cells were washed twice in ice cold sterile 15% glycerol. Cells were resuspended in 2 ml 15% glycerol and frozen in 50 μl aliquots using a dry ice-ethanol bath. Plasmid DNA was added to an aliquot of competent cells thawed on ice and then transferred to a 0.1 cm gap electroporation cuvette (BioRad). Cells were electroporated using a Gene Pulser II (BioRad) with the following settings: voltage, 1.8 kV; resistance, 200 Ω; capacitance, 25 μFd.

Experimental infections. Eight-week-old, specific-pathogen-free, castrated, male pigs (Whiteshire Hamroc, Inc., Albion, Ind.) were allotted to six challenge groups by a stratified random sampling procedure, balancing each group for body weight. Each challenge group was housed in a separate BSL-2 (biosafety level) isolation room at the Michigan State University Research Containment Facility. All experimental protocols for animal experiments were reviewed by the Michigan State University All University Committee on Animal Use and Care, and all procedures conformed to university and USDA regulations and guidelines.

For preparation of challenge inocula, bacteria were grown in 30 ml HIV+5 mM CaCl₂ +riboflavin and antibiotics as needed, in 300 ml baffled side-arm flasks, at 37° C. with shaking at 160 RPM, to an OD₅₂₀ (optical density) of 0.8. Ten ml of each culture was harvested by centrifugation at room temperature and washed once with sterile 0.9% saline. The cell pellet was resuspended in 10 mL of saline and diluted in saline to obtain the desired cfu/ml. The actual inoculating doses were retrospectively calculated by viable cell counts on agar plates.

For the challenge procedure, pigs were anesthetized by intravenous injection with ketamine (4.4 mg/kg) and xylazine (1.65 mg/kg) and inoculated by percutaneous intratracheal injection with the appropriate dose of bacteria suspended in 10 mL saline. Clinical signs of pleuropneumonia, including increased respiration rate, fever, dyspnea, decreased appetite and activity/attitude (depression), were monitored and scored as previously described (Jolie, R. A. V., M. H. Mulks, and B. J. Thacker. 1995. Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 45:383-391). Seriously ill animals, as determined by severe dyspnea and/or depression, were euthanized immediately. Survivors were euthanized three days post-challenge. All animals were necropsied, and lungs were examined macroscopically for A. pleuropneumoniae lesions, including edema, congestion, hemorrhage, necrosis, abscessation, fibrosis, and pleuritis. The percentage of lung tissue and pleural surface area affected was estimated for each of the seven lung lobes, and the total % pneumonia and % pleuritis calculated using a formula that weights the contribution of each lung lobe to the total lung volume (Jolie, R. A. V., M. H. Mulks, and B. J. Thacker. 1995. Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 45:383-391). Representative lung samples were collected for histopathology and for bacterial culture.

RESULTS

Construction of A. pleuropneumoniae rib mutants. To construct riboflavin-requiring auxotrophic mutants of A. pleuropneumoniae, a suicide plasmid with part of the riboflavin operon deleted and replaced with a kanamycin-resistance (Km^(R)) cassette was designed (FIG. 7). A 2.9 kb EcoRI fragment from pTF10 (Fuller, T. E. and M. H. Mulks. 1995. Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes. J. Bacteriol. 177:7265-7270) containing the A. pleuropneumoniae ribBAH genes was cloned into the EcoRI site of the conjugative suicide vector pGP704 (18) to create plasmid pTF66. pTF66 was digested with ClaI and NdeI to excise the 3' end of ribB and the entire ribA gene. After Klenow treatment of the DNA, the 1.2 kb Km^(R) cassette, excised with EcoRI from pUC4K , was blunt-end ligated into the rib deletion site to create pTF67a.

pTF67a was transformed into E. coli S17-1 (λpir) and mobilized into AP225 (Nal^(R)) to produce >100 transconjugant colonies demonstrating resistance to both nalidixic acid and kanamycin. Transconjugants were replica plated onto TSAV and TSAV+riboflavin to assess the requirement for riboflavin and the stability of the riboflavin auxotrophy. Two classes of transconjugants were found. The majority of the transconjugants, e.g. AP234, were unstable and produced revertants capable of growth without supplemental riboflavin in the absence of kanamycin selection. One transconjugant, AP233, was very stable, maintaining kanamycin resistance as well as the inability to grow without exogenous riboflavin. All transconjugants were confirmed as A. pleuropneumoniae by gram stain, colonial morphology, and requirement for V factor (β-NAD).

Southern blot analysis of transconjugants. Two transconjugants were selected for further analysis based on their phenotypes as potential single (AP234) and double cross-over mutants (AP233). Southern blot analysis of transconjugant genomic DNA from the two mutants indicated that AP233 and AP234 were indeed double and single cross-over insertion mutants respectively (FIG. 8A). Predicted band sizes for single and double cross-over events are shown in FIG. 8A. Genomic DNA from AP233 contained a 2.2 Kb HindIII fragment that hybridized with the riboflavin operon (Rib) probe, as well as 1.7 and 1.3 Kb fragments that hybridized with both the Rib and Km probes; however, there was no reaction with either pGP704 nor the deleted portion of the riboflavin operon (FIG. 8B). This is the pattern of hybridization predicted in transconjugants that replaced the wild type riboflavin operon with the mutated rib: :Km^(R) locus by a double-crossover event (FIG. 8A). In contrast, genomic DNA from AP234 shows the presence of DNA homologous to the fragment deleted from the riboflavin operon (R. del), pGP704, and the kanamycin cassette (FIG. 8B). This is the pattern of hybridization predicted in transconjugants that inserted the entire pTF67a plasmid into the wild type rib operon by a single crossover event (FIG. 8A).

Phenotypic analysis of the A. pleuropneumoniae rib mutant. Whole cell lysates, TCA-precipitated culture supernatants, and polysaccharide preparations were analyzed on silver stained SDS-PAGE and on immunoblots developed with convalescent swine sera. No differences in protein, LPS, extracellular toxin, or capsular polysaccharide profiles were detected between wild type AP100, its Nal^(R) derivative AP225, and the riboflavin mutant AP233 (data not shown). There was no difference in reactivity with serotype-specific antisera as determined by coagglutination assay (data not shown).

Complementation of the rib mutation with a cloned wild type rib operon. The 5.2 Kb insert from pTF10, containing the wild-type A. pleuropneumoniae riboflavin operon, was cloned into pGZRSl9, an E. coli-A. pleuropneumoniae shuttle vector (West, S. E. H., M. J. M. Romero, L. B. Regassa, N. A. Zielinski, and R. A. Welch. 1995. Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic resistance genes. Gene 160: 81-86), to form pTF76. pTF76 was transformed into AP233 by electroporation, restoring the ability of AP233 to grow in the absence of exogenous riboflavin and restoring the virulence of the mutant (see below).

Attenuation of virulence of the rib mutant in swine. Six groups of three pigs each were infected with: group 1, 1 LD₅₀ (5×10⁶ cfu) of AP225; groups 2-5, AP233 at doses equivalent to 4, 20, 100, and 500 times the wild-type LD₅₀ ; and group 6, 1 wild-type LD₅₀ of AP233/pTF76. Mortality, lung score, and clinical score data, shown in Tables 3, 4 and 5, all indicate that the riboflavin auxotroph is avirulent in pigs at doses as high as 500 times the wild-type LD₅₀. The pigs infected with the rib mutant AP233 displayed no dyspnea, elevated respiration rate, depression, or loss of appetite, and had no typical pleuropneumonic pathology at necropsy, at even the highest dose tested. In contrast, 1 of 3 pigs infected with the wild-type AP225 strain died, and all three exhibited significant clinical signs of APP disease, including elevated respiration rates, dyspnea, depression, loss of appetite, and fever, and severe pneumonia and pleuritis was evident at necropsy. Pigs infected with AP233 containing the riboflavin genes in trans (pTF76) also exhibited obvious clinical signs and significant pneumonia and pleuritis, although somewhat less severe than the wild-type strain. These results indicate that restoration of the ability to synthesize riboflavin does restore virulence.

Bacteria were readily reisolated at necropsy from the lungs of pigs receiving AP225 and AP233/pTF76. All reisolated organisms were characterized by gram stain, colonial morphology, requirement for V factor (β-NAD), antibiotic sensitivity, and serotyping by coagglutination. Reisolated organisms showed no differences from the initial inocula, including the presence of plasmid pTF76 in bacteria reisolated from pigs infected with AP233/pTF76. In contrast, we were unable to recover organisms from the lungs of animals infected with AP233 and euthanized 48 hours post infection.

                  TABLE 3     ______________________________________     Characteristics of bacterial strains and plasmids     Strain/                      Source/     Plasmid            Characteristics       Reference     ______________________________________     Strain     E. coli            supE44, ΔlacU169, (φ80lacZΔM15),                                  BRL (USA)     DH5-α            hsdR17, recA1, endA1, gyrA96, thi-1,            relA1     E. coli            λpir, supE44, ΔlacU169,                                  Mulks & Buysse     DH5-α            (φ80lacZΔM15), hsdR17, recA1, endA1,     (λpir)            gyrA96, thi-1, relA1     E. coli            λpir, recA, thi, pro, hsd, (r-m+),                                  Simon et al.     S17-1  RP4-2, (Tc::Mu), (Km::Tn7),  TmpR!,     (λpir)             SmR!     AP100  A. pleuropneumoniae ATCC 27088,                                  ATCC            serotype 1, passaged through pigs     AP106  A. pleuropneumoniae ISU178, a                                  Iowa State            serotype 5 field isolate, passaged                                  University            through pigs     AP225  A spontaneous nalidixic acid                                  This work            resistant mutant of AP100     AP233  A double cross-over riboflavin                                  This work            auxotroph of AP225     AP234  A single cross-over riboflavin                                  This work            auxotroph of AP225     Plasmid     pUC19  Ap.sup.R cloning vector                                  Vieira &                                  Messing     pUC4K  Ap.sup.R, Km.sup.R vector, source of the kan                                  Pharmacia            cassette              (USA)     pGP704 Ap.sup.R broad host range suicide vector                                  Miller &                                  Mekalanos     pGZRS19            Ap.sup.R APP-E. coli shuttle vector                                  West et al.     pTF10  AP106 ribGBAH genes cloned into                                  Fuller & Mulks            pUC19     pTF66  A 2.9 kb fragment containing AP106                                  This work            ribBAH in pGP704     pTF67a pTF66 with all of ribA and part of                                  This work            ribB deleted and replaced with the            kan cassette from pUC4K     pTF76  5.2 Kb insert from pTF10 cloned into                                  This work            pGZRS19     ______________________________________

                  TABLE 4     ______________________________________     Mortality and Lung Lesion Data                       Dose    Mor-  %       %     Group Strain      (LD.sub.50).sup.a                               tality                                     Pneumonia.sup.b                                             Pleuritis.sup.c     ______________________________________     1     AP225 (WT)  1       1/3   66.7    71.7     2     AP233 (Rib-)                       4       0/3   0       0     3     AP233 (Rib-)                       20      0/3   0       0     4     AP233 (Rib-)                       100     0/3   0       0     5     AP233 (Rib-)                       500     0/3   0       0     6     AP233 + pTF76                       1       0/3   27.6    20.2     ______________________________________      .sup.a Doses are multiples of the established wildtype APP 225serotype 1      LD.sub.50 of 5.0 × 10.sup.6 cfu (12)      .sup.b Percentage of lung tissue exhibiting A. pleuropneumoniae lesions      .sup.c Percentage of pleural surface area exhibiting pleuritis

                                      TABLE 5     __________________________________________________________________________     Clinical Score Data     Group          Strain   Dose (LD.sub.50).sup.a                           RR Max.sup.b                                 Temp Max.sup.c                                        Dyspnea.sup.d                                              Depression.sup.e                                                     Appetite.sup.f     __________________________________________________________________________     1    AP225    1       20    105.7  5.5   6.7    4.2     2    AP233    5       8     102.5  0     0      0     3    AP233    20      8     103.3  0     0      0     4    AP233    100     8     103.5  0     0      0     5    AP233    500     8     102.8  0     0      0     6    AP233 + pTF76                   1       19.3  105.4  4.5   4.7    3.7          Normal           8.0   <103.0 0     0      0          Maximum          25           15    15     5     __________________________________________________________________________      .sup.a Doses are multiples of the established wildtype APP225serotype 1      LD.sub.50 of 5.0 × 10.sup.6 cfu (12)      .sup.b Maximum respiratory rate observed after challenge. Respiratory rat      recorded as number of breaths per 15 sec observation period.      .sup.c Maximum rectal temperature after challenge, in degrees Fahrenheit.      .sup.d Dyspnea score measures degree of respiratory distress and labored      breathing. Scored as 0 = normal; 1 = slight; 2 = moderate; 3 = severe.      Total score = sum of scores taken at 12 hour intervals after challenge.      .sup.e Depression score evaluates attitude and activity. Scored as 0 =      normal; 1 = slight inactivity; 2 = moderate; 3 = severe. Total score = su      of scores taken at 12 hour intervals after challenge.      .sup.f Appetite was scored as 0 = did eat; 1 = did not eat. Total score =      number of 12 hour periods not eating over 60 hour observation period.

DISCUSSION

Above is shown the construction of a serotype 1 Actinobacillus pleuropneumoniae deletion-disruption riboflavin mutant that is attenuated in vivo. The A. pleuropneumoniae ribGBAH operon was disrupted by deleting an internal segment of the operon (ribBA) and replacing it with a Km^(R) cassette using a targeted mutagenesis technique (Mulks, M. H. and J. M. Buysse. 1995. A targeted mutagenesis system for Actinobacillus pleuropneumoniae. Gene 165:61-66). A stable riboflavin-requiring, Km^(R) mutant, AP233, was phenotypically identical to its wild-type parent based on analysis of proteins, extracellular toxin, LPS, and capsular polysaccharide by SDS-PAGE, immunoblot, and coagglutination.

A riboflavin mutant of A. pleuropneumoniae serotype 5 was also constructed and was also found to be attenuated in a preliminary animal challenge experiment. However, further studies were conducted in serotype 1 because serotype 5 seemed to be very resistant to transformation by standard heat shock or electroporation procedures. In order to complement the rib mutation in trans, and for ease of future genetic manipulations, it was desirable to use a serotype 1 strain for these studies.

Experimental infection of pigs, the only natural host for A. pleuropneumoniae, demonstrated that the riboflavin-requiring mutant was unable to cause disease at dosages as high as 500 times the LD₅₀ for the wild-type parent. In the four groups of pigs infected with AP233 by intratracheal inoculation, there was no mortality, no significant clinical signs were observed, and no typical pleuropneumonic lesions were observed at necropsy. Complementation of AP233 in trans with the wild-type A. pleuropneumoniae riboflavin operon restored both the ability to grow without exogenous riboflavin and virulence, demonstrating that the riboflavin mutation itself is responsible for the attenuation in vivo.

It is important to note that the riboflavin-requiring mutant used in these studies is a deletion mutant, with ˜1.4 Kb of the riboflavin operon removed from the chromosome and replaced with an antibiotic resistance marker. Neither reversion to prototrophy nor loss of kanamycin resistance in this mutant in the laboratory was observed. In the preliminary experiment with a serotype 5 riboflavin mutant, it was possible to reisolate the mutant from the lungs at 16 hours post-infection. All colonies isolated in this experiment were kanamycin-resistant, nalidixic acid-resistant, and riboflavin requiring, suggesting that reversion to prototrophy and thus virulence will not occur in vivo.

In the dosage trial experiment, AP233 was not recovered from the lungs of infected swine at 48 hours post-infection. These results may indicate poor persistence of the organism in vivo. If necessary, sufficient exogenous riboflavin could be added to the vaccine to allow the organism to replicate minimally and therefore persist long enough to induce a protective immune response. The above represents a new addition to the group of biosynthetic mutations that can be used to construct attenuated strains of bacteria. It also shows a genetically modified attenuated mutant of APP that is capable of production of all of the major virulence factors of this organism, including extracellular toxins and capsular polysaccharide.

EVALUATION OF A RIBOFLAVIN-REQUIRING AUXOTROPHIC MUTANT OF ACTINOBACILLUS PLEUROPNEUMONIAE AS A GENETICALLY DEFINED LIVE ATTENUATED VACCINE AGAINST PORCINE PLEUROPENUMONIA

The applicants have evaluated a genetically defined riboflavin-requiring attenuated mutant of Actinobacillus pleuropneumoniae as a live avirulent vaccine that provides immunity against experimental challenge with a virulent strain of A. pleuropneumoniae.

The specific aims of this study were: 1) to evaluate whether respiratory exposure to a live attenuated vaccine APP strain elicits protection against subsequent experimental challenge with virulent A. pleuropneumoniae; and 2) to determine whether addition of exogenous riboflavin to the vaccine dosage improves persistence, and therefore immunogenicity and protection; and 3) to compare the protection afforded by respiratory exposure to that elicited by intramuscular (IM) immunization with the live vaccine, which is a more commercially feasible vaccination route.

MATERIALS AND METHODS

Animals. In this study, 6-to-8 week old crossbred (Yorkshire/Landrace) barrows from a herd known to be free of A. pleuropneumoniae and related respiratory pathogens were used. Pigs were housed in the Michigan State University Research Containment Facility and fed a standard antibiotic-free diet provided by the MSU Swine Research and Teaching Center.

Preparation of Vaccines.

1. Live vaccine: The bacterial strain used to prepare the live attenuated vaccine was AP233, a derivative of the species type strain, ATCC27088 (here designated APP-1A) that is resistant to nalidixic acid (Nal^(R)), resistant to kanamycin (Kan^(R)), and that requires riboflavin (Rib-) because it contains a riboflavin biosynthetic operon that has been mutated by deletion-disruption with a kanamycin resistance cassette. Bacteria for the live vaccine were grown in heart infusion broth containing 10 μg/ml NAD (nicotine adenine dinucleotide)+5 mM CaCl₂ +200 μg/ml riboflavin, at 37 C., to an optical density at 520 nm of 0.8. Bacteria were harvested, washed once in phosphate buffered saline (PBS), pH 7.0, diluted in phosphate buffered saline (PBS) or PBS containing 5 μg/ml riboflavin to the appropriate cell density, and used immediately as vaccine.

2. Bacterin: Virulent APP-1A bacteria were grown in heart infusion broth containing 10 μg/ml NAD (nicotine adenine dinucleotide)+5 mM CaCl₂ at 37 C., shaking at 160 rpm, to an optical density at 520 nm of 0.8. Bacteria were harvested by centrifugation and washed once with Tris-acetate-EDTA-DTT buffer. Bacteria were resuspended in buffer containing 0.2% formalin to a concentration of 5×10⁹ cfu/ml, and kept at room temperature for 1 hour, then stored at 4° C. Each vaccine dose contained 1 ml formalinized cells, 0.5 ml saline, and 0.5 ml Emulsigen adjuvant (MVP Laboratories, Ralston, Nebr.).

Vaccine groups. There were six treatment groups (six pigs/group) in this study. Pigs were blocked by starting weight and randomly assigned to treatment groups. The animals were vaccinated twice at a 3 week interval, and challenged with virulent APP serotype 1A (APP-1) two weeks after the second vaccination. Group 1 received 5×10⁸ cfu (100× the 50% lethal dose previously established for the wild type parent strain WT LD₅₀ !) of live AP233, our APP-1 riboflavin-requiring mutant, in 10 ml of sterile PBS, by percutaneous transtracheal inoculation, as in our challenge model (described below). Group 2 received the same treatment as Group 1, except the bacteria were suspended in 10 ml of PBS containing 5 μg/ml riboflavin, a concentration of exogenous riboflavin sufficient to permit 2-3 generations of growth. Group 3 received 5×10⁸ cfu of live AP233, intramuscularly in 2 ml PBS. Group 4 received the same treatment as Group 3, except the bacteria were suspended in PBS plus 5 μg/ml riboflavin. Group 5 received a formalinized whole cell bacterin prepared from APP-1, which contained the equivalent of 5×10⁹ cfu per dose, in 2 ml of 25% Emulsigen adjuvant (MVP Laboratories, Ralston, Nebr.). Group 6 were unvaccinated controls.

Experimental challenge. Two weeks after the second vaccination, all groups of pigs were challenged with virulent wild type APP-1A, using an experimental challenge model (Jolie, R. A. V., M. H. Mulks, and B. J. Thacker. 1995. Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 45: 383-391; Thacker, B. J., M. H. Mulks, B. Yamini, & J. Krehbiel. 1988. Clinical, immunological, hematological, microbiological, and pathological evaluation of a percutaneous intratracheal injection Haemophilus pleuropneumoniae challenge model. Proc. Int. Pig Vet. Soc. 10: 69). For the challenge inoculum, bacteria were grown to late exponential phase in heart infusion broth containing 10 μg/ml NAD+5 mM CaCl₂, washed once in sterile saline, and diluted in saline to the appropriate cell density. Pigs were anesthetized by intravenous injection with a mixture of ketamine (6.6 mg/kg) and xylazine (1.65 mg/kg) and inoculated transtracheally with 1 LD₅₀ (5×10⁶ cfu) of APP-1 suspended in 10 ml saline. Clinical signs, including increased rectal temperature, increased respiration rate, dyspnea, decreased appetite, and depression, were monitored at 4 hour intervals for the first 24 hours post infection, and at 12 hour intervals thereafter. Severely ill animals, as determined by the severity of clinical signs, were euthanized by overdose with a pentobarbital solution (Beuthanasia) delivered intravenously and necropsied immediately. Three days post-infection, all surviving pigs were euthanized and necropsied, and gross pathology of the lungs examined and compared. Lungs were examined macroscopically for APP lesions, including edema, congestion, hemorrhage, infarction, necrosis, abscess, fibrosis, and pleuritis. The percentage of lung tissue and surface area affected was estimated for each of the seven lung lobes, and the data inserted into a formula that weights the contribution of each lung lobe to give a total percentage of lung involvement and affected pleural surface (Thacker, B. J., M. H. Mulks, B. Yamini, & J. Krehbiel. 1988. Clinical, immunological, hematological, microbiological, and pathological evaluation of a percutaneous intratracheal injection Haemophilus pleuropneumoniae challenge model. Proc. Int. Pig Vet. Soc. 10: 69). Tissue samples were collected and processed for histopathology, and for culture of APP to confirm infection. Protection of pigs against challenge was measured as a reduction in mortality, in the severity of lung lesions, and in the severity and duration of clinical signs as compared to the unvaccinated control animals. Statistical analysis of the data was conducted using the Statistix microcomputer program (Analytical Software, Tallahassee, Fla.) for analysis of variance (ANOVA) and Epistat (T. L. Gustafson, Round Rock, Tex.) for nonparametric analyses.

RESULTS

Safety. Pigs were monitored post-vaccination for any clinical signs of APP disease, such as fever, dyspnea, and increased respiratory rate, and for injection site reactions in Group 3, 4, and 5 animals. The bacterin vaccinated animals (Group 5) showed mild fever, depression, and decrease in appetite for 8-16 hours post-vaccination, which is a common reaction to bacterin vaccines. Several of the Group 5 animals had granulomatous reactions at the injection site in the neck muscle, which were detected at necropsy. The Group 1 and 2 animals, which received intratracheal immunizations, showed increased respiratory rates, fever, decreased appetite, and mild depression for 8-16 hours post-immunization. The Group 3 and 4 animals, which received the intramuscular vaccine, showed only slight depression and decreased appetite for <8 hours, and no significant fever or increase in respiratory rate. No injection site reactions were detected in the Group 3 or 4 animals at necropsy. These results demonstrate that the live intramuscular vaccine is at least as safe as, if not safer than, a formalinized bacterin of the type routinely used commercially at this time.

Immunogenicity. The immune responses of the pigs to vaccination were evaluated by ELISA against APP outer membranes (Jolie, R. A. V., M. H. Mulks, and B. J. Thacker. 1995. Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B. Vet. Microbiol. 45: 383-391); by hemolysin neutralization titer (Montaraz, J. A., B. Fenwick, H. Hill, and M. Rider. 1996. Evaluating antibody isotype-specific ELISA, complement fixation, and ApxI hemolysin neutralization tests to detect serum antibodies in pigs infected with Actinobacillus pleuropneumoniae serotype 1. Swine Health and Production 4: 79-83); and by complement fixation (CF) (Hoffman, L. J. 1989. Actinobacillus (Haemophilus) pleuropneumoniae: Use of coagglutination and complement fixation to determine the relationship between presence of organisms and antibody titer in slaughterhouse pigs. J. Vet. Diagn. Invest. 1:12-15)(Table 1).

At challenge, the bacterin-vaccinated animals showed significant ELISA and complement fixation titers, but low or negative hemolysin neutralization titers. The four groups receiving live vaccines showed low or negative ELISA and CF titers. However, the Group 3 and 4 animals did show significant hemolysin neutralization titers.

                  TABLE 6     ______________________________________     Serologic analysis of serum samples collected at challenge     Group #           Vaccine   HN.sup.1    ELISA-APP1.sup.2                                          CF.sup.3     ______________________________________     1     Live, IT,  3129 ± 1478.sup.b                                  227 ± 90.sup.b                                           1.7 ± 2.8.sup.b           PBS     2     Live, IT,  2520 ± 741.sup.b                                  164 ± 73.sup.b,c                                           1.6 ± 3.1.sup.b           PBS +           riboflavin     3     Live, IM, 10760 ± 6245.sup.a                                  120 ± 32.sup.b,c                                           0.0 ± 0.0.sup.b           PBS     4     Live, IM,  6293 ± 2662.sup.a,b                                  236 ± 173.sup.b                                           2.0 ± 4.0.sup.b           PBS +           riboflavin     5     APP-1A     3035 ± 285.sup.b                                 1119 ± 170.sup.a                                          24.3 ± 7.4.sup.a           bacterin     6     Unvac-     2240 ± 243.sup.b                                  67 ± 21.sup.c                                           0.0 ± 0.0.sup.b           cinated           control     ______________________________________      .sup.1 Hemolysin neutralization titer; <3000 = negative; 3000-6000 =      suspect; >6000 = positive. Assays performed in the laboratory of Dr. Brad      Fenwick, Kansas State University.      .sup.2 ELISA vs APP1 outer membranes; <200 = negative, 200-300 = suspect,      >300 = positive. Assays performed in the laboratory of Dr. Martha H.      Mulks, Michigan State University.      .sup.3 Complement fixation test; reported as geometric mean titer 0 =      negative; >0 = positive. Assays performed at the Veterinary Diagnostic      Laboratory, Iowa State University.

Addition of riboflavin to the inoculum. In preliminary studies, it was found that riboflavin-requiring strains of APP failed to persist in the porcine respiratory tract for more than 16-24 hours. Poor persistence of live vaccine strains in vivo can lead to a failure to elicit a protective immune response. A. pleuropneumoniae and other related pathogens can produce infection-associated antigens when grown in an appropriate host. These are antigens that are only produced by the bacterium when it is grown within a host animal, presumably due to specific environmental stimuli such as temperature, lack of available iron, pH, or osmotic conditions (Mekalanos, J. J. 1992. Environmental signals controlling expression of virulence determinants in bacteria. Infect. Immun. 174:1-7.). Such infection associated antigens are not produced when the bacterium is grown in vitro in standard laboratory media. In order to assure that such infection-associated antigens would be expressed by the live attenuated vaccine strain of bacteria after immunization of pigs, it was necessary to ensure that the bacteria had sufficient available riboflavin to permit 2-3 generations of growth. It was determined that addition of 5-10 μg of riboflavin per ml of the vaccine inoculum was sufficient to permit this amount of growth. Therefore, as part of this vaccine trial, intratracheal (IT) and intramuscular (IM) administration of the live attenuated vaccine, with and without the addition of 5 μg/ml exogenous riboflavin, were compared.

Riboflavin may be added to permit two generations of growth such that the amount may vary from about 1 to about 10 μg/ml.

Protection against challenge. In this experiment, the live attenuated vaccine prepared with exogenous riboflavin and delivered intramuscularly (Group 4) provided complete protection against mortality (0/5 animals died) and a significant reduction in lung damage and in some clinical signs of pleuropneumonia (Tables 2 and 3). In contrast, 6/6 unvaccinated control animals died from overwhelming pleuropneumonia as a result of this experimental challenge. Other live vaccine formulations, as well as the formalinized bacterin, afforded less protection than the intramuscular immunization containing riboflavin. It is concluded that 1) intramuscular immunization with this live vaccine does elicit significant protection against APP infection; 2) that intratracheal immunization does not elicit the same degree of protection; and 3) that the addition of exogenous riboflavin improves the efficacy of the live vaccine.

Lung cultures. APP was cultured from the lungs of all the challenged pigs except for 1 animal in Group 4. All cultures were confirmed as APP-1A by gram stain, requirement for NAD, and coagglutination.

                  TABLE 7     ______________________________________     Mortality and Lung Score Data     Group Vaccine.sup.1                        Mortality                                 % Pneumonia.sup.2                                          % Pleuritis.sup.3     ______________________________________     1     Live, IT, PBS                        3/5      58.6 ± 23.5.sup.a,b                                          73.3 ± 39.3.sup.a     2     Live, IT, PBS +                        6/6      63.2 ± 8.2.sup.a,b                                          66.7 ± 51.6.sup.a           riboflavin     3     Live, IM, PBS                        4/6      57.7 ± 23.2.sup.b                                          73.3 ± 42.5.sup.a     4     Live, IM, PBS +                        0/5      24.5 ± 15.0.sup.c                                          21.5 ± 20.7.sup.a           riboflavin     5     APP-1A bacterin                        3/6      54.1 ± 24.8.sup.b                                          73.9 ± 41.2.sup.a     6     Unvaccinated 6/6      80.9 ± 13.2.sup.a                                          83.3 ± 40.8.sup.a           control     ______________________________________      .sup.1 IT: live vaccine administered by intratracheal inoculation; IM:      live vaccine administered by intramuscular injection.      .sup.2 Percentage of lung tissue exhibiting A. pleuropneumoniae lesions;      results presented as mean ± standard deviation.      .sup.3 Percentage of pleural surface area exhibiting pleuritis; results      presented as mean ± standard deviation.      .sup.a . . . c Values with different superscripts among the six vaccine      groups were significantly different (p < 0.05) by Least Significant      Difference (LSD) analysis.

                                      TABLE 8     __________________________________________________________________________     Clinical Score Data     Group          Vaccine.sup.1                     RR Max.sup.2                            Temp Max.sup.3                                   Dyspnea.sup.4                                          Depression.sup.5                                                 Appetite.sup.6     __________________________________________________________________________     1    Live, IT, PBS                     22.0 ± 5.2.sup.a                             104.7.sup.a                                   1.80 ± .45.sup.a,b                                          1.40 ± .55.sup.a                                                 2.00 ± .71.sup.a     2    Live, IT, PBS +                     19.7 ± 5.7.sup.a                             104.0.sup.a                                   2.17 ± .41.sup.a                                          1.67 ± .82.sup.a                                                 0.75 ± .96.sup.b,c          riboflavin     3    Live, IM, PBS                     19.2 ± 1.2.sup.a                             104.7.sup.a                                   1.83 ± 41.sup.a,b                                          1.27 ± .75.sup.a                                                 2.33 ± 1.21.sup.a     4    Live, IM, PBS +                     18.2 ± 3.4.sup.a                             104.2.sup.a                                   1.20 ± .75.sup.b                                          0.40 ± .89.sup.a                                                 0.20 ± .45.sup.c          riboflavin     5    APP-1A bacterin                     23.3 ± 1.6.sup.a                             104.7.sup.a                                   1.83 ± 52.sup.a,b                                          1.83 ± .75.sup.a                                                 1.67 ± 1.03.sup.a,b     6    Unvaccinated control                     23.0 ± 5.8.sup.a                             104.9.sup.a                                   2.33 ± .52.sup.a                                          1.83 ± .75.sup.a                                                 1.83 ± .90.sup.a,b          Normal      8.0   <103.0 0      0      0          Maximum    25            3      3      3     __________________________________________________________________________      .sup.1 IT: live vaccine administered by intratracheal inoculation; IM:      live vaccine administered by intramuscular injection.      .sup.2 Maximum respiratory rate observed after challenge. Respiratory rat      recorded as number of breaths per 15 second observation period.      .sup.3 Maximum rectal temperature after challenge, in degrees Fahrenheit.      .sup.4 Maximum dyspnea score observed after challenge. Dyspnea score      measures degree of respiratory distress and labored breathing. Scored as      = normal; 1 = slight; 2 = moderate; 3 = severe.      .sup.5 Maximum depression score observed after challenge. Depression scor      evaluates attitude and activity. Scored as 0 = normal; 1 = slight      inactivity; 2 = moderate; 3 = severe.      .sup.6 Appetite was scored as 0 = did eat; 1 = did not eat. Total score =      number of 12 hour periods not eating over 36 hour observation period.      .sup.a . . . c Values with different superscripts among the six vaccine      groups were significantly different (p < 0.05) by Least Significant      Difference (LSD) analysis.

It is concluded that intramuscular vaccination with the live attenuated riboflavin-requiring A. pleuropneumoniae mutant, with the addition of a limited amount of exogenous riboflavin, led to complete protection against mortality and to significant reduction in lung damage and clinical signs of pleuropneumonia.

The applicants have determined that other related species of pathogenic bacteria in the Family Pasteurellaceae contain homologous genes encoding riboflavin biosynthetic enzymes. Southern blot analysis of genomic DNA from Actinobacillus suis and Pasteurella haemolytica demonstrated that these species contain DNA fragments that are highly homologous to the rib genes from A. pleuropneumoniae. Genomic DNA from these species was digested with the restriction endonucleases EcoRI and HindIII, fragments separated on an agarose gel, and the fragments transferred to nitrocellulose. The nitrocellulose blot was probed with a digoxigenin-labelled probe prepared from the ribGBAH operon from APP serotype 5, at 42° C., in a hybridization cocktail that included 50% formamide, 5×SSC (20×SSC contains 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.1% N-lauroylsarcosine, and 0.02% SDS. The blot was washed under high stringency conditions, including two 15 minute washes at room temperature in 2×SSC, 0.1% SDS, followed by two 30 minute washes at 68° C. in 0.1×SSC, 0.1% SDS. P. haemolytica contains an ˜12 kb DNA HindIII fragment that hybridized with the rib probe, while A. suis contains three EcoRI fragments of ˜4.4, 2.5, and 1.0 kb that are highly homologous to the APP-5 rib probe. These data suggest that these species of bacteria contain riboflavin operons that are similar to that analyzed from APP serotype 5.

A live avirulent vaccine against APP is desirable. There are a variety of different kinds of vaccines produced to elicit protection against bacterial diseases. Some of the most effective are purified toxins converted to toxoids. These toxoid vaccines are often very safe, and can be very effective against diseases where a toxin is the major virulence factor. Examples would be current vaccines against tetanus and diphtheria. These vaccines do not prevent acquisition and carriage of the causative organism, e.g, Clostridium tetani, the causative agent of tetanus, or Corynebacterium diphtheriae, the agent of diphtheria. Rather, they prevent the deleterious effects of the toxin by eliciting antibodies that neutralize the toxin. In other cases where a key virulence factor has been identified, purified protein or polysaccharide vaccines have been produced. Examples here would be the E. coli pilin vaccine against porcine colibacillosis and the capsular polysaccharide vaccines now available against Haemophilus influenzae B, Streptococcus pneumoniae, and some serotypes of Neisseria meningitidis. These vaccines either prevent initial adherence of the pathogen, as in the case of the pilin vaccine, or enhance phagocytosis and clearance of the pathogen, as in the case of the capsular polysaccharide vaccines. In the veterinary field, it is also common to use bacterin vaccines, that is, killed whole cell vaccines. Because these bacterins can induce a wide range of side effects, they are not commonly used for human vaccines. A problem with all of these types of vaccines is that they generally induce systemic humoral immunity, i.e., serum antibodies. It is difficult to induce local secretory immunity with these types of vaccines. Live avirulent vaccines, where the recipient of the vaccine receives a dose of infectious but not virulent bacteria, can be an improvement over purified subunit or killed whole cell vaccines, for several reasons. First, the vaccine dose can often be administered to the same region of the body that is normally infected by the pathogen, e.g., orally for a gastrointestinal pathogen or as a nasal spray for a respiratory pathogen, This can elicit local secretory immunity as well as systemic humoral immunity. Second, live avirulent vaccines can often be administered as a single dose rather than multiple doses, because the organism can continue to grow and replicate within the host, providing a longer term exposure to important antigens that a single dose of killed vaccine. Finally, live avirulent vaccines may provide exposure to important bacterial antigens not contained in killed vaccines grown in the laboratory. For example, if a bacterium produces important antigens or virulence factors whose expression is induced by in vivo environmental signals, these antigens may not be contained in a vaccine prepared from bacteria grown in vitro in laboratory media. It is desirable for a vaccine to elicit cross-protective immunity against the different serotypes of APP. It is known that vaccination with a killed whole cell vaccine prepared from a single serotype of APP will usually not elicit cross-protective immunity against other serotypes. However, infection with a virulent strain of APP will generally elicit at least some degree of cross-protection against other serotypes. One explanation for this phenomenon is that antigens may be expressed by APP during growth in vivo that elicit a cross-protective immune response, and that these antigens are not contained in most bacteria vaccines.

Specifically, it has been shown that extracellular toxins, referred to as hemolysins/cytolysins, are produced by APP in vivo but are not produced under the culture conditions typically used for producing killed whole cell vaccines.

The applicants have shown that riboflavin-requiring mutants of APP can be effective as a live avirulent vaccine. There are two basic methods for producing live avirulent vaccine strains. One is to knock out a critical virulence factor necessary for survival in vivo and perhaps also for disease/damage to the host. An example would be Inzana's non-capsulated APP mutants. These mutants are unable to synthesize capsular polysaccharide, which acts in vivo to protect the bacterium from phagocytosis and clearance by alveolar macrophages. Non-capsulated mutants simply can not survive long enough in vivo to cause disease. They do, however, presumably express all the other important virulence factors and therefore should elicit an immune response against antigens other than capsular polysaccharide.

A second method to produce live avirulent vaccines is to knock out genes in biosynthetic pathways known to be critical for survival in vivo. For example, the availability of compounds such as purines and aromatic amino acids is limited in mammalian hosts. Bacterial pathogens must be able to synthesize these compound themselves, or scavenge them from host tissues. Mutations in the biosynthetic pathways for purines and aromatic amino acids have been used to construct bacterial mutants that can not survive long in vivo, and thus have potential for use as attenuated vaccines. Much of the current research on genetically engineered live avirulent vaccines has been done with members of the genus Salmonella. These studies show that purA mutants are avirulent but poorly immunogenic (O'Callaghan et al, 1988), while mutations in the chorismate pathway, including aroA, aroc, and aroD, are attenuated and can be effective as live oral vaccines (Doggett & Curtiss, 1992; Tacket et al, 1992). In addition, Salmonella strains carrying cya and crp mutations, which produce mutants that lack the enzyme adenylate cyclase and the cyclic AMP receptor protein, which are required for the expression of numerous critical genes in bacteria, have been shown to be both avirulent and immunogenic (Doggett & Curtiss, 1992; Tacket et al, 1992; Kelly et al, 1992).

Riboflavin is an essential vitamin and biosynthetic precursor for the coenzymes FMN and FAD. It is synthesized by most bacteria, but not by mammals. Therefore, it is expected that riboflavin would be in limited supply in a mammalian host and that a bacterium incapable of synthesizing its own riboflavin would be attenuated. This has been shown above. It has also been shown above that Rib- mutants can survive long enough in the host to be immunogenic and effective as a live avirulent vaccine.

The rib-APP mutant may be combined with a sterile, buffered, isotonic, pharmaceutically-acceptable and compatible aqueous carrier such as saline, or saline derivative such as citrate-buffered saline, tris-buffered saline, Ringer's Solution or tissue culture medium, and the like, preferably having a physiologic pH. An antigen composition may also include a suitable compatible adjuvant such as aluminum hydroxide, paraffin-based oils, averdine, muramyl dipeptide, and the like, to stabilize the antigen in solution, and/or an immunomodulator such as a recombinant cytokine or interleukin such as IL-1, IL-5, IL-6, TGF-beta, or gamma interferon, and the like, to enhance the IgA antibody response. However, the adjuvant chosen should not contain any preservative, such as formalin, that would be deleterious to a live vaccine. In the experiments described above, no adjuvant was used.

The vaccine composition may be formulated for administration as a single injection of about 0.5 to 10 ml. The composition may also be in the form for administration in a series of biweekly or monthly injections of about 0.5 to 10 ml each, until the desired level of immunity is achieved. Preferably,the composition is formulated for a single administration to the animal.

The vaccine composition as described herein may be formulated with conventional pharmaceutically acceptable vehicles for administration by transthoracic intrapulmonary injection, intratracheal innoculation, subcutaneous, intraperitoneal or intramuscular injection. The vaccine may also be supplied orally or intranasally. These vehicles comprise substances that are essentially nontoxic and nontherapeutic such as saline and derivatives of saline such as citrate-buffered saline, tris-buffered saline and Ringer's Solution, dextrose solution, Hank's Solution, tissue culture medium, and the like. The antigen composition may also include minor but effective amounts of pharmaceutically-accepted adjuvants, buffers and preservatives to maintain isotonicity, physiological pH, and stability. Adjuvants useful in the composition include, but are not limited to, for example, paraffin based oils, averdine, muramyl dipeptide, and oil-in-water-based adjuvants, andthe like. Examples of suitable buffers include but not limited to, phosphate buffers, citrate buffers, carbonate buffers, TRIS buffers, and the like. It is also envisioned that the antigen may be combined with a biocompatible, and optimally synergistic, immunomodulator that cooperatively stimulates IgA antibody production, as for example, but not limited to, recombinant cytokines such as TGF-beta, interferons, activating factors, chemoattractants, interleukins such as IL-1, IL-2, IL-4, IL-5, IL-6 and the like, and other like substances.

While the forms of the invention herein disclosed constitute presently preferred embodiments, many others are possible. It is not intended here to mention all the possible equivalent forms or ramifications of the invention. It is understood that the terms used herein are merely descriptive, and that various changes may be made without departing from the spirit or scope of the invention.

FIGURE LEGENDS

FIG. 1. Proposed bacterial riboflavin biosynthesis pathway. Proposed gene functions are as indicated although the functions of ribG and ribT have not been determined conclusively. Structures correspond to the following: I, GTP; II, 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone 5'-phosphate; III, 5-amino-6-(ribosylamino)-2,4(1H,3H)-pyrimidinedione 5'-phosphate; IV, 5-amino-6-(ribitylamino)-2,4(1H,3H)-pyrimidinedione 5'-phosphate; V, 5-amino-6-(ribitylamino)-2,4(1H,3H) -pyrimidinedione; VI, ribulose 5'-phosphate; VII, 3,4-dihydroxy-2-butanone 4-phosphate; VIII, 6,7-dimethyl-8-ribityllumazine; IX, riboflavin. Structures are adapted from Bacher (1).

FIG. 2. Physical map of the construct, pTF10, which contains the APP riboflavin synthesis genes.

FIG. 3. Absorbance spectra of aqueous solutions at neutral pH (Panel A) and acidified aqueous solutions (Panel B) of the product excreted into the growth medium by E. coli DH5α/pTF10 (solid line) and of a standard riboflavin preparation (dotted line).

FIG. 4. Complete nucleotide sequence of APP ribGBAH genes and flanking regions. The amino acid translations are shown for ribG, ribB, ribA, and ribH and correspond to base pairs 330-1560, 1685-2330, 2393-3596 and 3709-4168. Putative ribosome binding sites are underlined. Potential promoters for the operon and for ribH are double-underlined. An inverted repeat that may function as a transcription terminator is indicated with arrows.

FIG. 5. Complementation of E. coli mutants by cloned APP rib genes. A physical map for the APP ribGBAH genes is shown as well as several deletions that were made from the 3' end of the APP rib clone. The E. coli gene designations are indicated above their APP homologues. A "+" indicates complementation of the indicated E. coli mutation by the recombinant plasmid. nd=not done.

FIG. 6. Minicell analysis of pTF10 and deletions. Minicells contained: Lane 1, pUC19; Lane 2, pTF10; Lane 3, pTF19. Molecular weight standards are indicated on the left. Proteins encoded by the APP genes are indicated by the arrows on the right. Apparent molecular weights for the APP Rib enzymes are: RibG, 45 kDa; RibA, 43.7 kDa; RibB, 27.7 kDa, and RibH, 14.8 kDa.

FIG. 7. Construction of pTF67A. The entire riboflavin operon, containing the ribGBAH genes from AP106 was cloned into pUC19 to make pTF10 (5). A 2.9 kb fragment containing the ribBAHportion of the riboflavin operon was excised from pTF10 with EcoRI and ligated into the EcoRI site of the conjugative suicide vector pGP704 to form pTF66. A 1.4 kb ClaI/NdeI fragment, which contains all of ribA and part of ribb was deleted and replaced with the Km^(R) cassette from pUC4K to create pTF67a.

FIG. 8. Southern blot analysis of chromosomal DNA from AP100 rib mutants.

(A) Schematic structure of the rib locus of parent and mutant strains in double and single cross-over events. The predicted sizes of HindIII genomic fragments are shown for two possible single cross-over events and for a double cross-over event. The results show that for AP233 the chromosomal rib operon has been replaced with the cloned riboflavin operon containing the Km^(R) cassette by a double cross-over event, while AP234 is the result of a single cross-over event either upstream or downstream of the kanamycin cassette. Restriction enzymes used: E=EcoRI; H=HindIII.

(B) Southern blots of HindIII or EcoRI digested DNA from mutants and controls. Blots were prepared in quadruplicate and hybridized at high stringency with one of four probes: Rib, the entire ribGBAH operon from pTF10; R. Del., the deleted portion (ClaI/NdeI fragments) of the ribGBAH operon; pGP704, the entire plasmid; Km, the kanamycin cassette from pUC4K. Lanes: 1, pTF10 digested with HindIII; 2, AP106+HindIII; 3, AP100+HindIII; 4, pTF67a+EcoRI; 5, AP233+HindIII; and 6, AP234+HindIII.

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    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES:17     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:21 bases               (B) TYPE:nucleic acid               (C) STRANDEDNESS:single               (D) TOPOLOGY:linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     #21                AAGG C     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH: 19 bases               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     # 19               ACG     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:20 bases               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     - 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#l Leu Val Arg Asn Gly     #   140     - His Thr Glu Ala Thr Val Asp Leu Ala Arg Le - #u Ala Gly Leu Lys His     145                 1 - #50                 1 - #55                 1 -     #60     - Ala Gly Leu Cys Cys Glu Ile Met Ala Asp As - #p Gly Thr Met Met Thr     #               175     - Met Pro Asp Leu Gln Lys Phe Ala Val Glu Hi - #s Asn Met Pro Phe Ile     #           190     - Thr Ile Gln Gln Leu Gln Glu Tyr Arg Arg Ly - #s His Asp Ser Leu Val     #       205     - Lys Gln Ile Ser Val Val Lys Met Pro Thr Ly - #s Tyr Gly Glu Phe Met     #   220     - Ala His Ser Phe Val Glu Val Ile Ser Gly Ly - #s Glu His Val Ala Leu     225                 2 - #30                 2 - #35                 2 -     #40     - Val Lys Gly Asp Leu Thr Asp Gly Glu Gln Va - #l Leu Ala Arg Ile His     #               255     - Ser Glu Cys Leu Thr Gly Asp Ala Phe Gly Se - #r Gln Arg Cys Asp Cys     #           270     - Gly Gln Gln Phe Ala Ala Ala Met Thr Gln Il - #e Glu Gln Glu Gly Arg     #       285     - Gly Val Ile Leu Tyr Leu Arg Gln Glu Gly Ar - #g Gly Ile Gly Leu Ile     #   300     - Asn Lys Leu Arg Ala Tyr Glu Leu Gln Asp Ly - #s Gly Met Asp Thr Val     305                 3 - #10                 3 - #15                 3 -     #20     - Glu Ala Asn Val Ala Leu Gly Phe Lys Glu As - #p Glu Arg Glu Tyr Tyr     #               335     - Ile Gly Ala Gln Met Phe Gln Gln Leu Gly Va - #l Lys Ser Ile Arg Leu     #           350     - Leu Thr Asn Asn Pro Ala Lys Ile Glu Gly Le - #u Lys Glu Gln Gly Leu     #       365     - Asn Ile Val Ala Arg Glu Pro Ile Ile Val Gl - #u Pro Asn Lys Asn Asp     #   380     - Ile Asp Tyr Leu Lys Val Lys Gln Ile Lys Me - #t Gly His Met Phe Asn     385                 3 - #90                 3 - #95                 4 -     #00     - Phe     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH: 645 bases               (B) TYPE: nucleic acid               (C) STRANDEDNESS:single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     #              50TATTGA AGAAGTCGGC AAAATTGCTC AAATTCATAA     #             100CGGTAG TCACAATTAA TGCGACCAAA GTATTACAAG     #             150GACACG ATTGCGGTGA ACGGCGTATG TTTAACCGTA     #             200TAATCA GTTTACCGCC GATGTAATGT CGGAAACGTT     #             250TAGGCG AATTAAAGTC GAATAGTCCG GTTAATTTAG     #             300GCAAAC GGACGTTTCG GCGGACACAT CGTTTCGGGG     #             350CGGCGA AATTGCGGAA ATCACACCGG CACATAATTC     #             400TTAAAA CCTCTCCAAA ATTAATGCGT TATATTATTG     #             450ACCATT GACGGTATTA GCCTGACCGT AGTCGATACC     #             500CCGTGT ATCGATTATT CCGCATACGA TTAAAGAAAC     #             550AAAAAA TCGGCAGTAT TGTCAATTTA GAAAATGATA     #             600ATCGAA CAGTTTTTAC TGAAAAAGCC GGCGGATGAG     #                 645TT AGACTTTTTA AAGCAGGCGG GATTT     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 215 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     - Met Phe Thr Gly Ile Ile Glu Glu Val Gly Ly - #s Ile Ala Gln Ile His     #                 15     - Lys Gln Gly Glu Phe Ala Val Val Thr Ile As - #n Ala Thr Lys Val Leu     #             30     - Gln Asp Val His Leu Gly Asp Thr Ile Ala Va - #l Asn Gly Val Cys Leu     #         45     - Thr Val Thr Ser Phe Ser Ser Asn Gln Phe Th - #r Ala Asp Val Met Ser     #     60     - Glu Thr Leu Lys Arg Thr Ser Leu Gly Glu Le - #u Lys Ser Asn Ser Pro     # 80     - Val Asn Leu Glu Arg Ala Met Ala Ala Asn Gl - #y Arg Phe Gly Gly His     #                 95     - Ile Val Ser Gly His Ile Asp Gly Thr Gly Gl - #u Ile Ala Glu Ile Thr     #           110     - Pro Ala His Asn Ser Thr Trp Tyr Arg Ile Ly - #s Thr Ser Pro Lys Leu     #       125     - Met Arg Tyr Ile Ile Glu Lys Gly Ser Ile Th - #r Ile Asp Gly Ile Ser     #   140     - Leu Thr Val Val Asp Thr Asp Asp Glu Ser Ph - #e Arg Val Ser Ile Ile     145                 1 - #50                 1 - #55                 1 -     #60     - Pro His Thr Ile Lys Glu Thr Asn Leu Gly Se - #r Lys Lys Ile Gly Ser     #               175     - Ile Val Asn Leu Glu Asn Asp Ile Val Gly Ly - #s Tyr Ile Glu Gln Phe     #           190     - Leu Leu Lys Lys Pro Ala Asp Glu Pro Lys Se - #r Asn Leu Ser Leu Asp     #       205     - Phe Leu Lys Gln Ala Gly Phe     #   215     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:1230 bases               (B) TYPE:nucleic acid               (C) STRANDEDNESS:single               (D) TOPOLOGY:linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     #              50TAAGCG GTGGTTTTTC CTATCTTTTT TACAAGCCTT     #             100TCAAGG CTTTTTTCAT CATTAGGGTA AACATGCCTG     #             150CTGCCC TCAAATAGTT TCAAAACAAT GACGGATTTA     #             200TGCCAT TGCACTGGCA AAACAAGGTT TAGGCTGGAC     #             250TTGTCG GTTGTGTAAT TGTCAAAAAC GGTGAAATCG     #             300CATGAA AAGATTGGTG GATGGCATGC GGAACGTAAT     #             350TAAGGA AGATCTTTCC GGGGCGACTG CTTATGTAAC     #             400GTCATC ACGGCCGCAC GCCGCCTTGT TCGGATTTAT     #             450ATTAAA AAAGTATTTA TCGGTTCGAG CGATCCGAAT     #             500GCGGGG AGCAAATCAG CTACGCCAAG CCGGCGTGGA     #             550TACTCA AAGAAGAATG TGATGCGTTA AACCCGATTT     #             600CAAACT AAACGTCCGT ATGTGCTAAT GAAATATGCC     #             650CAAAAT TGCAACCGGT AGCGGCGAAT CCAAATGGAT     #             700CAAGAG CAAGAGTGCA GCAAACACGT CATCAATATA     #             750GGTGTA GATACGGTAC TTGCCGATAA CCCGATGTTA     #             800GAATGC GAAACAACCG GTCCGGATTG TCTGCGATAG     #             850CGTTAG ATTGCCAGTT AGTGCAGACA GCGAAAGAAT     #             900GCAACC GTTAGTGACG ATTTGCAAAA AATTGAACAA     #             950CGTAGA TGTATTAGTG TGTAAAGCAC GAAACAAGCG     #            1000ATCTTT TGCAAAAGCT CGGTGAAATG CAGATCGACA     #            1050GGCGGT TCAAGTTTGA ATTTCAGTGC GTTAGAAAGC     #            1100AGTACA TTGTTATATT GCGCCTAAAT TAGTCGGTGG     #            1150CCCCAA TCGGCGGTGA GGGAATTCAA CAAATCGACC     #            1200AAATTG AAATCGACCG AACTCATCGG CGAAGATATT     #         1230     TCAT CTCCCCTCTT     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 410 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     - Met Lys Leu Pro Cys Lys Arg Trp Phe Phe Le - #u Ser Phe Leu Gln Ala     #                 15     - Leu Arg Ser Lys Asp Phe Lys Ala Phe Phe Il - #e Ile Arg Val Asn Met     #             30     - Pro Val Met Cys Phe Pro Leu Pro Ser Asn Se - #r Phe Lys Thr Met Thr     #         45     - Asp Leu Asp Tyr Met Arg Arg Ala Ile Ala Le - #u Ala Lys Gln Gly Leu     #     60     - Gly Trp Thr Asn Pro Asn Pro Leu Val Gly Cy - #s Val Ile Val Lys Asn     # 80     - Gly Glu Ile Val Ala Glu Gly Tyr His Glu Ly - #s Ile Gly Gly Trp His     #                 95     - Ala Glu Arg Asn Ala Val Leu His Cys Lys Gl - #u Asp Leu Ser Gly Ala     #           110     - Thr Ala Tyr Val Thr Leu Glu Pro Cys Cys Hi - #s His Gly Arg Thr Pro     #       125     - Pro Cys Ser Asp Leu Leu Ile Glu Arg Gly Il - #e Lys Lys Val Phe Ile     #   140     - Gly Ser Ser Asp Pro Asn Pro Leu Val Ala Gl - #y Arg Gly Ala Asn Gln     145                 1 - #50                 1 - #55                 1 -     #60     - Leu Arg Gln Ala Gly Val Glu Val Val Glu Gl - #y Leu Leu Lys Glu Glu     #               175     - Cys Asp Ala Leu Asn Pro Ile Phe Phe His Ty - #r Ile Gln Thr Lys Arg     #           190     - Pro Tyr Val Leu Met Lys Tyr Ala Met Thr Al - #a Asp Gly Lys Ile Ala     #       205     - Thr Gly Ser Gly Glu Ser Lys Trp Ile Thr Gl - #y Glu Ser Ala Arg Ala     #   220     - Arg Val Gln Gln Thr Arg His Gln Tyr Ser Al - #a Ile Met Val Gly Val     225                 2 - #30                 2 - #35                 2 -     #40     - Asp Thr Val Leu Ala Asp Asn Pro Met Leu As - #n Ser Arg Met Pro Asn     #               255     - Ala Lys Gln Pro Val Arg Ile Val Cys Asp Se - #r Gln Leu Arg Thr Pro     #           270     - Leu Asp Cys Gln Leu Val Gln Thr Ala Lys Gl - #u Tyr Arg Thr Val Ile     #       285     - Ala Thr Val Ser Asp Asp Leu Gln Lys Ile Gl - #u Gln Phe Arg Pro Leu     #   300     - Gly Val Asp Val Leu Val Cys Lys Ala Arg As - #n Lys Arg Val Asp Leu     305                 3 - #10                 3 - #15                 3 -     #20     - Gln Asp Leu Leu Gln Lys Leu Gly Glu Met Gl - #n Ile Asp Ser Leu Leu     #               335     - Leu Glu Gly Gly Ser Ser Leu Asn Phe Ser Al - #a Leu Glu Ser Gly Ile     #           350     - Val Asn Arg Val His Cys Tyr Ile Ala Pro Ly - #s Leu Val Gly Gly Lys     #       365     - Gln Ala Lys Thr Pro Ile Gly Gly Glu Gly Il - #e Gln Gln Ile Asp Gln     #   380     - Ala Val Lys Leu Lys Leu Lys Ser Thr Glu Le - #u Ile Gly Glu Asp Ile     385                 3 - #90                 3 - #95                 4 -     #00     - Leu Leu Asp Tyr Val Val Ile Ser Pro Leu     #               410     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:459 bases               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     #              50AGGTAA CTTAGTTGCG ACAGGTTTAA AATTCGGTAT     #             100TCAACG ATTTTATCAA CGATAAATTA TTAAGCGGTG     #             150GTGCGT CACGGTGCGT ATGAAAACGA TATTGATACG     #             200TGCATT TGAGATTCCA TTAGTTGCGA AAAAAATGGC     #             250ATGATG CGGTAATCTG TTTAGGTACG GTAATTCGCG     #             300TATGAT TACGTATGTA ATGAAGCGGC AAAAGGTATC     #             350AGAAAC CGGCGTACCG GTAATTTTCG GTGTATTAAC     #             400AACAGG CGATTGAACG CGCGGGTACT AAAGCAGGTA     #             450TGTGCA TTAGGCGCAA TCGAAATAGT AAACGTATTA     #        459     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 153 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     - Met Ala Lys Ile Thr Gly Asn Leu Val Ala Th - #r Gly Leu Lys Phe Gly     #                 15     - Ile Val Thr Ala Arg Phe Asn Asp Phe Ile As - #n Asp Lys Leu Leu Ser     #             30     - Gly Ala Ile Asp Thr Leu Val Arg His Gly Al - #a Tyr Glu Asn Asp Ile     #         45     - Asp Thr Ala Trp Val Pro Gly Ala Phe Glu Il - #e Pro Leu Val Ala Lys     #     60     - Lys Met Ala Asn Ser Gly Lys Tyr Asp Ala Va - #l Ile Cys Leu Gly Thr     # 80     - Val Ile Arg Gly Ser Thr Thr His Tyr Asp Ty - #r Val Cys Asn Glu Ala     #                 95     - Ala Lys Gly Ile Gly Ala Val Ala Leu Glu Th - #r Gly Val Pro Val Ile     #           110     - Phe Gly Val Leu Thr Thr Glu Asn Ile Glu Gl - #n Ala Ile Glu Arg Ala     #       125     - Gly Thr Lys Ala Gly Asn Lys Gly Ser Glu Cy - #s Ala Leu Gly Ala Ile     #   140     - Glu Ile Val Asn Val Leu Lys Ala Ile     145                 1 - #50     __________________________________________________________________________ 

What is claimed is:
 1. A vaccine against Actinobacillus pleuropneumoniae (APP) comprising a recombinant APP lacking a gene necessary for producing riboflavin and a pharmaceutically acceptable carrier.
 2. The vaccine of claim 2 wherein the gene lacking from APP is one of the rib genes.
 3. The vaccine of claim 3 wherein the rib gene is selected from the group consisting of rib G, rib A, rib B and rib H.
 4. A method of vaccinating a mammal in need thereof comprising administering to the mammal an effective vaccinating amount of the vaccine of claim
 1. 5. A method of stimulating the immune system of a mammal in need thereof comprising the steps of:a. providing a recombinant APP lacking a gene necessary for producing riboflavin; b. administering an effective immunogenic amount of the recombinant APP in a pharmaceutically acceptable carrier to a mammal, in need thereof, thereby causing an antigenic response thereto in the mammal.
 6. A method of inducing protective immunity in a mammal in need thereof against disease caused by APP comprising the step of administering to the mammal an effective amount of recombinant APP lacking a gene necessary for producing riboflavin such that the recombinant APP causes protective immunity in the mammal against APP.
 7. The method of claim 5 wherein the mammal is a pig.
 8. The method of claim 6 wherein the mammal is a pig. 